Compositions for chromosome-specific staining

ABSTRACT

Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods are provided to disable the hybridization capacity of shared, high copy repetitive sequences and/or remove such sequences to provide for useful contrast. Still further methods are provided to produce chromosome-specific staining reagents which are made specific to the targeted chromosomal material, which can be one or more whole chromosomes, one or more regions on one or more chromosomes, subsets of chromosomes and/or the entire genome. Probes and test kits are provided for use in tumor cytogenetics, in the detection of disease related loci, in analysis of structural abnormalities, such as translocations, and for biological dosimetry. Further, methods and prenatal test kits are provided to stain targeted chromosomal material of fetal cells, including fetal cells obtained from maternal blood. Still further, the invention provides for automated means to detect and analyse chromosomal abnormalities.

The United States Government has rights in this invention pursuant toContract No. W-7405-ENG-48 between the U.S. Department of Energy and theUniversity of California, for the operation of Lawrence LivermoreNational Laboratory.

RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 08/242,075, filedMay 13, 1994, now abandoned, which is a continuation of U.S. Ser. No.08/120,190, filed Sep. 13, 1993, now abandoned, which is a continuationof U.S. Ser. No. 07/862,060, filed Apr. 2, 1992, now abandoned, which isa continuation of U.S. Ser. No. 07/444,669, filed Dec. 1, 1989, nowabandoned, which is a continuation in part of U.S. Ser. No. 937,793,filed Dec. 4, 1986, now abandoned which is, in turn, a continuation inpart of U.S. Ser. No. 819,314, filed Jan. 16, 1986 now abandoned by thenamed inventors hereof and assigned to the same assignee and claimspriority in said prior filed applications.

FIELD OF THE INVENTION

The invention relates generally to the field of cytogenetics, and moreparticularly, to the field of molecular cytogenetics. The inventionconcerns methods for identifying and classifying chromosomes. Still moreparticularly, this invention concerns nucleic acid probes which can bedesigned by the processes described herein to produce stainingdistributions that can extend along one or more whole chromosomes,and/or along a region or regions on one or more chromosomes, includingstaining patterns that extend over the whole genome. Staining patternscan be tailored for any desired cytogenetic application, includingprenatal, tumor and disease related cytogenetic applications, amongothers. The invention provides for compositions of nucleic acid probesand for methods of staining chromosomes therewith to identify normalchromosomes and chromosomal abnormalities in metaphase spreads and ininterphase nuclei. The probe-produced staining patterns of thisinvention facilitate the microscopic and/or flow cytometricidentification of normal and abnormal chromosomes and thecharacterization of the genetic nature of particular abnormalities.

Although most of the examples herein concern human chromosomes and muchof the language herein is directed to human concerns, the concept ofusing nucleic acid probes for staining or painting chromosomes isapplicable to chromosomes from any source including both plants andanimals.

BACKGROUND OF THE INVENTION

Chromosome abnormalities are associated with genetic disorders,degenerative diseases, and exposure to agents known to causedegenerative diseases, particularly cancer, German, "Studying HumanChromosomes Today," American Scientist, Vol. 58, pgs. 182-201 (1970);Yunis, "The Chromosomal Basis of Human Neoplasia," Science, Vol. 221,pgs. 227-236 (1983); and German, "Clinical Implication of ChromosomeBreakage," in Genetic Damage in Man Caused by Environmental Agents,Berg, Ed., pgs. 65-86 (Academic Press, New York, 1979). Chromosomalabnormalities can be of several types, including: extra or missingindividual chromosomes, extra or missing portions of a chromosome(segmental duplications or deletions), breaks, rings and chromosomalrearrangements, among others. Chromosomal rearrangements includetranslocations (transfer of a piece from one chromosome onto anotherchromosome), dicentrics (chromosomes with two centromeres), andinversions (reversal in polarity of a chromosomal segment).

Detectable chromosomal abnormalities occur with a frequency of one inevery 250 human births. Abnormalities that involve deletions oradditions of chromosomal material alter the gene balance of an organismand generally lead to fetal death or to serious mental and physicaldefects. Down syndrome can be caused by having three copies ofchromosome 21 instead of the normal 2. This syndrome is an example of acondition caused by abnormal chromosome number, or aneuploidy. Downsyndrome can also be caused by a segmental duplication of a subregion onchromosome 21 (such as, 21q22), which can be present on chromosome 21 oron another chromosome. Edward syndrome (18+), Patau syndrome (13+),Turner syndrome (XO) and Kleinfelter syndrome (XXY) are among the mostcommon numerical aberrations. Epstein, The Consequences of ChromosomeImbalance: Principles, Mechanisms and Models (Cambridge Univ. Press1986); Jacobs, Am. J. Epidemiol, 105:180 (1977); and Lubs et al.,Science, 1:495 (1970).!

Retinoblastoma (del 13q14), Prader-Willi syndrome (del 15q11→q13),Wilm's tumor (del 11p13) and Cri-du-chat syndrome (del 5p) are examplesof important disease linked structural aberrations. Nora and Fraser,Medical Genetics: Principles and Practice, (Lea and Febiger 1989).!

Chronic myelogeneous leukemia is associated with the exchange ofchromosomal material between chromosome 9 and chromosome 22. Thetransfer of chromosomal material in this leukemia is an example of atranslocation. Clearly, a sensitive method for detecting chromosomalabnormalities would be a highly useful tool for genetic screening.

Measures of the frequency of structurally aberrant chromosomes, forexample, dicentric chromosomes, caused by clastogenic agents, such as,ionizing radiation or chemical mutagens, are widely used as quantitativeindicators of genetic damage caused by such agents, BiochemicalIndicators of Radiation Injury in Man (International Atomic EnergyAgency, Vienna, 1971); and Berg, Ed. Genetic Damage in Man Caused byEnvironmental Agents (Academic Press, New York, 1979). A host ofpotentially carcinogenic and teratogenic chemicals are widelydistributed in the environment because of industrial and agriculturalactivity. These chemicals include pesticides, and a range of industrialwastes and by-products, such as halogenated hydrocarbons, vinylchloride, benzene, arsenic, and the like, Kraybill et al., Eds.,Environmental Cancer (Hemisphere Publishing Corporation, New York,1977). Sensitive measures of chromosomal breaks and other abnormalitiescould form the basis of improved dosimetric and risk assessmentmethodologies for evaluating the consequences of exposure to suchoccupational and environmental agents.

Current procedures for genetic screening and biological dosimetryinvolve the analysis of karyotypes. A karyotype is the particularchromosome complement of an individual or of a related group ofindividuals, as defined both by the number and morphology of thechromosomes usually in mitotic metaphase. It includes such things astotal chromosome number, copy number of individual chromosome types(e.g., the number of copies of chromosome X), and chromosomalmorphology, e.g., as measured by length, centromeric index,connectedness, or the like. Chromosomal abnormalities can be detected byexamination of karyotypes. Karyotypes are conventionally determined bystaining an organism's metaphase, or otherwise condensed (for example,by premature chromosome condensation) chromosomes. Condensed chromosomesare used because, until recently, it has not been possible to visualizeinterphase chromosomes due to their dispersed condition and the lack ofvisible boundaries between them in the cell nucleus.

A number of cytological techniques based upon chemical stains have beendeveloped which produce longitudinal patterns on condensed chromosomesgenerally referred to as bands. The banding pattern of each chromosomewithin an organism usually permits unambiguous identification of eachchromosome type, Latt, "Optical Studies of Metaphase ChromosomeOrganization," Annual Review of Biophysics and Bioengineering, Vol. 5,pgs. 1-37 (1976). Accurate detection of some important chromosomalabnormalities, such as translocations and inversions, has required suchbanding analysis.

Unfortunately, such conventional banding analysis requires cellculturing and preparation of high quality metaphase spreads, which istime consuming and labor intensive, and frequently difficult orimpossible. For example, cells from many tumor types are difficult toculture, and it is not clear that the cultured cells are representativeof the original tumor cell population. Fetal cells capable of beingcultured need to be obtained by invasive means and need to be culturedfor several weeks to obtain enough metaphase cells for analysis. In manycases, the banding patterns on the abnormal chromosomes do not permitunambiguous identification of the portions of the normal chromosomesthat make them up. Such identification may be important to indicate thelocation of important genes involved in the abnormality. Further, thesensitivity and resolving power of current methods of karyotyping arelimited by the fact that multiple chromosomes or chromosomal regionshave highly similar staining characteristics, and that abnormalities(such as deletions) which involve only a fraction of a band are notdetectable. Therefore, such methods are substantially limited for thediagnosis and detailed analysis of contiguous gene syndromes, such aspartial trisomy, Prader-Willi syndrome Emanuel, Am. J. Hum. Genet.,43:575 (1988); Schmickel, J. Pediatr., 109:231 (1986)! andretinoblastoma Sparkes, Biochem. Biophys, Acta., 780:95 (1985)!.

Thus, conventional banding analysis has several important limitations,which include the following. 1) It is labor intensive, time consuming,and requires a highly trained analyst. 2) It can be applied only tocondensed chromosomes. 3) It does not allow for the detection ofstructural aberrations involving less than 3-15 mega-bases (Mb),depending upon the nature of the aberration and the resolution of thebanding technique Landegren et al., Science, 242:229 (1988)!. Thisinvention provides for probe compositions and methods to overcome suchlimitations of conventional banding analysis.

The chemical staining procedures of the prior art provide patterns overa genome for reasons not well understood and which cannot be modified asrequired for use in different applications. Such chemical stainingpatterns were used to map the binding site of probes.

However, only occasionally, and with great effort, was in situhybridization used to obtain some information about the position of alesion, for example, a breakpoint relative to a particular DNA sequence.The present invention overcomes the inflexibility of chemical stainingin that it stains a genome in a pattern based upon nucleic acidsequence; therefore the pattern can be altered as required by changingthe nucleic acid sequence of the probe. The probe-produced stainingpatterns of this invention provide reliable fundamental landmarks whichare useful in cytogenetic analysis.

Automated detection of structural abnormalities of chromosomes withimage analysis of chemically stained bands would require the developmentof a system that can detect and interpret the banding patterns producedon metaphase chromosomes by conventional techniques. It has proven to bevery difficult to identify reliably by automated means normalchromosomes that have been chemically stained; it is much more difficultto differentiate abnormal chromosomes having structural abnormalities,such as, translocations. Effective automated detection of translocationsin conventionally banded chromosomes has not been accomplished afterover a decade of intensive work. The probe-produced banding patterns ofthis invention are suitable for such automated detection and analysis.

In recent years rapid advances have taken place in the study ofchromosome structure and its relation to genetic content and DNAcomposition. In part, the progress has come in the form of improvedmethods of gene mapping based on the availability of large quantities ofpure DNA and RNA fragments for probes produced by genetic engineeringtechniques, e.g., Kao, "Somatic Cell Genetics and Gene Mapping,"International Review of Cytology, Vol. 85, pgs. 109-146 (1983), andD'Eustachio et al., "Somatic Cell Genetics in Gene Families," Science,Vol. 220, pgs. 9, 19-924 (1983). The probes for gene mapping compriselabeled fragments of single-stranded or double-stranded DNA or RNA whichare hybridized to complementary sites on chromosomal DNA. With suchprobes it has been crucially important to produce pure, or homogeneous,probes to minimize hybridizations at locations other than at the site ofinterest, Henderson, "Cytological Hybridization to MammalianChromosomes," International Review of Cytology, Vol. 76, pgs. 1-46(1982).

The hybridization process involves unravelling, or melting, thedouble-stranded nucleic acids of the probe and target by heating, orother means (unless the probe and target are single-stranded nucleicacids). This step is sometimes referred to as denaturing the nucleicacid.

When the mixture of probe and target nucleic acids cool, strands havingcomplementary bases recombine, or anneal. When a probe anneals with atarget nucleic acid, the probe's location on the target can be detectedby a label carried by the probe or by some intrinsic characteristics ofthe probe or probe-target duplex. When the target nucleic acid remainsin its natural biological setting, e.g., DNA in chromosomes, mRNA incytoplasm, portions of chromsomes or cell nuclei (albeit fixed oraltered by preparative techniques), the hybridization process isreferred to as in situ hybridization.

In situ hybridization probes were initially limited to identifying thelocation of genes or other well defined nucleic acid sequences onchromosomes or in cells. Comparisons of the mapping of single-copyprobes to normal and abnormal chromosomes were used to examinechromosomal abnormalities. Cannizzaro et al., Cytogenetics and CellGenetics, 39:173-178 (1985). Distribution of the multiple binding sitesof repetitive probes could also be determined.

Hybridization with probes which have one target site in a haploidgenome, single-copy or unique sequence probes, has been used to map thelocations of particular genes in the genome Harper and Saunders,"Localization of the Human Insulin Gene to the Distal End of the ShortArm of Chromosome 11," Proc. Natl. Acad. Sci., Vol. 78, pgs. 4458-4460(1981); Kao et al., "Assignment of the Structural Gene Coding forAlbumin to Chromosome 4," Human Genetics, Vol. 62, pgs. 337-341 (1982)!;but such hybridizations are not reliable when the size of the targetsite is small. As the amount of target sequence for low complexitysingle-copy probes is small, only a portion of the potential targetsites in a population of cells form hybrids with the probe. Therefore,mapping the location of the specific binding site of the probe has beencomplicated by background signals produced by non-specific binding ofthe probe and also by noise in the detection system (for example,autoradiography or immunochemistry). The unreliability of signals forsuch prior art single-copy probes has required statistical analysis ofthe positions of apparent hybridization signals in multiple cells to mapthe specific binding site of the probe.

Wallace et al., in "The Use of Synthetic Oligonucleotides asHybridization Probes. II.

Hybridization of Oligonucleotides of Mixed Sequence to RabbitBeta-Globin DNA," Nucleic Acids Research, Vol. 9, pgs. 879-894 (1981),disclose the construction of synthetic oligonucleotide probes havingmixed base sequences for detecting a single locus corresponding to astructural gene. The mixture of base sequences was determined byconsidering all possible nucleotide sequences which could code for aselected sequence of amino acids in the protein to which the structuralgene corresponded.

Olsen et al., in "Isolation of Unique Sequence Human X ChromosomalDeoxyribonucleic Acid," Biochemistry, Vol. 19, pgs. 2419-2428 (1980),disclose a method for isolating labeled unique sequence human Xchromosomal DNA by successive hybridizations: first, total genomic humanDNA against itself so that a unique sequence DNA fraction can beisolated; second, the isolated unique sequence human DNA fractionagainst mouse DNA so that homologous mouse/human sequences are removed;and finally, the unique sequence human DNA not homologous to mouseagainst the total genomic DNA of a human/mouse hybrid whose only humanchromosome is chromosome X, so that a fraction of unique sequence Xchromosomal DNA is isolated. Individual clones are then isolated fromthis fraction and are candidates for human X chromosome specific DNAsequences.

Manuelidis et al., in "Chromosomal and Nuclear Distribution of the HindIII 1.9-KB Human DNA Repeat Segment," Chromosoma, Vol. 91, pgs. 28-38(1984), disclose the construction of a single kind of DNA probe fordetecting multiple loci on chromosomes corresponding to the location ofmembers of a family of repeated DNA sequences. Such probes are hereintermed repetitive probes.

Different repetitive sequences may have different distributions onchromosomes. They may be spread over all chromosomes as in the justcited reference, or they may be concentrated in compact regions of thegenome, such as, on the centromeres of the chromosomes, or they may haveother distributions. In some cases, such a repetitive sequence ispredominantly located on a single chromosome, and therefore is achromosome-specific repetitive sequence. Willard et al., "Isolation andCharacterization of a Major Tandem Repeat Family from the Human XChromosome," Nucleic Acids Research, Vol. 11, pgs. 2017-2033 (1983).!

A probe for repetitive sequences shared by all chromosomes can be usedto discriminate between chromosomes of different species if the sequenceis specific to one of the species. Total genomic DNA from one specieswhich is rich in such repetitive sequences can be used in this manner.Pinkel et al. (III), PNAS U.S.A., 83:2934 (1986); Manuelidis, Hum.Genet., 71: 288 (1985) and Durnam et al., Somatic Cell Molec. Genet.,11: 571 (1985.!

Recently, there has been an increased availability of probes forrepeated sequences (repetitive probes) that hybridize intensely andspecifically to selected chromosomes. Trask et al., Hum. Genet., 78: 251(1988) and references cited therein.! Such probes are now available forover half of the human chromosomes. In general, they bind to repeatedsequences on compact regions of the target chromosome near thecentromere. However, one probe has been reported that hybridizes tohuman chromosome 1p36, and there are several probes that hybridize tohuman chromosome Yq. Hybridization with such probes permits rapididentification of chromosomes in metaphase spreads, determination of thenumber of copies of selected chromosomes in interphase nuclei Pinkel etal. (I), PNAS U.S.A. 83: 2934 (1986); Pinkel et al. (II), Cold SpringHarbor Symp. Quant. Biol., 51: 151 (1986) and Cremer et al., Hum. Genet.74: 346 (1986)! and determination of the relative positions ofchromosomes in interphase nuclei Trask et al., supra; Pinkel et al. (I),supra; Pinkel et al. (II), supra; Manuelidis, PNAS U.S.A., 81: 3123(1984); Rappold et al., Hum. Genet., 67:317 (1984); Schardin et al.,Hum. Genet., 71: 282 (1985); and Manuelidis, Hum. Genet., 71: 288(1985)!.

However, many applications are still limited by the lack of appropriateprobes. For example, until the methods described herein were invented,probes with sufficient specificity for prenatal diagnosis were notavailable for chromosome 13 or 21. Further, repetitive probes are notvery useful for detection of structural aberrations since theprobability is low that the aberrations will involve the region to whichthe probe hybridizes.

This invention overcomes the prior art limitations on the use of probesand dramatically enhances the application of in situ hybridization forcytogenetic analysis. As indicated above, prior art probes have not beenuseful for in-depth cytogenetic analysis. Low complexity single-copyprobes do not at this stage of hybridization technology generatereliable signals. Although repetitive probes do provide reliablesignals, such signals cannot be tailored for different applicationsbecause of the fixed distribution of repetitive sequences in a genome.The probes of this invention combine the hybridization reliability ofrepetitive probes with the flexibility of being able to tailor thebinding pattern of the probe to any desired application.

The enhanced capabilities of the probes of this invention come fromtheir increased complexity. Increasing the complexity of a probeincreases the probability, and therefore the intensity, of hybridizationto the target region, but also increases the probability of non-specifichybridizations resulting in background signals. However, within theconcept of this invention, it was considered that such backgroundsignals would be distributed approximately randomly over the genome.Therefore, the net result is that the target region could be visualizedwith increased contrast against such background signals.

Exemplified herein are probes in an approximate complexity range of fromabout 50,000 bases (50 kb) to hundreds of millions of bases. Suchrepresentative probes are for compact loci and whole human chromosomes.Prior to this invention, probes employed for in situ hybridizationtechniques had complexities below 40 kb, and more typically on the orderof a few kb.

Staining chromosomal material with the probes of this invention issignificantly different from the chemical staining of the prior art. Thespecificity of the probe produced staining of this invention arises froman entirely new source--the nucleic acid sequences in a genome. Thus,staining patterns of this invention can be designed to highlightfundamental genetic information important to particular applications.

The procedures of this invention to construct probes of any desiredspecificity provide significant advances in a broad spectrum ofcytogenetic studies. The analysis can be carried out on metaphasechromosomes and interphase nuclei. The techniques of this invention canbe especially advantageous for applications where high-quality bandingby conventional methods is difficult or suspected of yielding biasedinformation, e.g., in tumor cytogenetics. Reagents targeted to sites oflesions known to be diagnostically or prognostically important, such astumor type-specific translocations and deletions, permit rapidrecognition of such abnormalities. Where speed of analysis is thepredominant concern, e.g., detection of low-frequency chromosomalaberrations induced by toxic environmental agents, the compositions ofthis invention permit a dramatic increase in detection efficiency incomparison to previous techniques based on conventional chromosomebanding.

Further, prenatal screening for disease-linked chromosome aberrations(e.g., trisomy 21) is enhanced by the rapid detection of suchaberrations by the methods and compositions of this invention.Interphase aneuploidy analysis according to this invention isparticularly significant for prenatal diagnosis in that it yields morerapid results than are available by cell culture methods. Further, fetalcells separated from maternal blood, which cannot be cultured by routineprocedures and therefore cannot be analysed by conventional karyotypingtechniques, can be examined by the methods and compositions of thisinvention. In addition, the intensity, contrast and color combinationsof the staining patterns, coupled with the ability to tailor thepatterns for particular applications, enhance the opportunities forautomated cytogenetic analysis, for example, by flow cytometry orcomputerized microscopy and image analysis.

SUMMARY OF THE INVENTION

This invention concerns methods of staining chromosomal material basedupon nucleic acid sequence that employ one or more nucleic acid probes.Said methods produce staining patterns that can be tailored for specificcytogenetic analyses. It is further an object of this invention toproduce nucleic acid probes that are useful for cytogenetic analysis,that stain chromosomal material with reliable signals. Such probes areappropriate for in situ hybridization. Preferred nucleic acid probes forcertain applications of this invention are those of sufficientcomplexity to stain reliably each of two or more target sites.

The invention provides methods and compositions for staining chromosomalmaterial. The probe compositions of this invention at the current stateof hybridization techniques are typically of high complexity, usuallygreater than about 50 kb of complexity, the complexity depending uponthe application for which the probe is designed. In particular,chromosome specific staining reagents are provided which compriseheterogeneous mixtures of nucleic acid fragments, each fragment having asubstantial fraction of its sequences substantially complementary to aportion of the nucleic acid for which specific staining is desired--thetarget nucleic acid, preferably the target chromosomal material. Ingeneral, the nucleic acid fragments are labeled by means as exemplifiedherein and indicated infra. However, the nucleic acid fragments need notbe directly labeled in order for the binding of probe fragments to thetarget to be detected; for example, such nucleic acid binding can bedetected by anti-RNA/DNA duplex antibodies and antibodies to thymidinedimers. The nucleic acid fragments of the heterogenous mixtures includedouble-stranded or single-stranded RNA or DNA.

One way to produce a probe of high complexity is to pool several or manyclones, for example, phage, plasmid, cosmid, and/or YAC clones, amongothers, wherein each clone contains an insert that is capable orhybridizing to some part of the target in a genome. Another way toproduce such a probe is to use the polymerase chain reaction (PCR).

Heterogeneous in reference to the mixture of labeled nucleic acidfragments means that the staining reagents comprise many copies each offragments having different sequences and/or sizes (e.g., from thedifferent DNA clones pooled to make the probe). In preparation for use,these fragments may be cut, randomly or specifically, to adjust the sizedistribution of the pieces of nucleic acid participating in thehybridization reaction.

As discussed more fully below, preferably the heterogeneous probemixtures are substantially free from nucleic acid sequences withhybridization capacity to non-target nucleic acid. Most of suchsequences bind to repetitive sequences which are shared by the targetand non-target nucleic acids, that is, shared repetitive sequences.

Methods to remove undesirable nucleic acid sequences and/or to disablethe hybridization capacity of such sequences are discussed more fullybelow. See Section II!. Such methods include but are not limited to theselective removal or screening of shared repetitive sequences from theprobe; careful selection of nucleic acid sequences for inclusion in theprobe; blocking shared repetitive sequences by the addition of unlabeledgenomic DNA, or, more carefully selecting nucleic acid sequences forinclusion in the blocking mixture; incubating the probe mixture forsufficient time for reassociation of high copy repetitive sequences, orthe like.

Preferably, the staining reagents of the invention are applied tointerphase or metaphase chromosomal DNA by in situ hybridization, andthe chromosomes are identified or classified, i.e., karyotyped, bydetecting the presence of the label, such as biotin or ³ H, on thenucleic acid fragments comprising the staining reagent.

The invention includes chromosome staining reagents for the totalgenomic complement of chromosomes, staining reagents specific to singlechromosomes, staining reagents specific to subsets of chromosomes, andstaining reagents specific to subregions within single or multiplechromosomes. The term "chromosome-specific," is understood to encompassall of these embodiments of the staining reagents of the invention. Theterm is also understood to encompass staining reagents made from anddirected against both normal and abnormal chromosome types.

A preferred method of making the chromosome-specific staining reagentsof the invention includes: 1) isolating chromosomal DNA from aparticular chromosome type or target region or regions in the genome, 2)amplifying the isolated DNA to form a heterogeneous mixture of nucleicacid fragments, 3) disabling the hybridization capacity of or removingshared repeated sequences in the nucleic acid fragments, and 4) labelingthe nucleic acid fragments to form a heterogeneous mixture of labelednucleic acid fragments. As described more fully below, the ordering ofthe steps for particular embodiments varies according to the particularmeans adopted for carrying out the steps.

The present invention addresses problems associated with karyotypingchromosomes, especially for diagnostic and dosimetric applications. Inparticular, the invention overcomes problems which arise because of thelack of stains that are sufficiently chromosome-specific by providingreagents comprising heterogeneous mixtures of nucleic acid fragmentsthat can be hybridized to the target DNA and/or RNA., e.g., the targetchromosomes, target subsets of chromosomes, or target regions ofspecific chromosomes. The staining technique of the invention opens upthe possibility of rapid and highly sensitive detection of chromosomalabnormalities in both metaphase and interphase cells using standardclinical and laboratory equipment and improved analysis using automatedtechniques. It has direct application in genetic screening, cancerdiagnosis, and biological dosimetry.

This invention further specifically provides for methods and nucleicacid probes for staining fetal chromosomal material, whether condensed,as in metaphase, or dispersed as in interphase. Still further, theinvention provides for a non-embryo-invasive method of karyotyping thechromosomal material of fetal cells, wherein the fetal cells have beenseparated from maternal blood. Such fetal cells are preferablyleukocytes and/or cytotrophoblasts. Exemplary nucleic acid probes arehigh complexity probes chromosome-specific for chromosome types 13, 18and/or 21. Representative probes comprise chromosome-specific Bluescribeplasmid libraries from which a sufficient number of shared repetitivesequences have been removed or the hybridization capacity thereof hasbeen disabled prior to and/or during hybridization with the target fetalchromosomes.

This invention still further provides for test kits comprisingappropriate nucleic acid probes for use in tumor cytogenetics, in thedetection of disease related loci, in the analysis of structuralabnormalities, for example translocations, and for biological dosimetry.

This invention further provides for prenatal screening kits comprisingappropriate nucleic acid probes of this invention.

The methods and compositions of this invention permit staining ofchromosomal material with patterns appropriate for a desiredapplication. The pattern may extend over some regions of one or morechromosomes, or over some or all the chromosomes of a genome and maycomprise multiple distinguishable sections, distinguishable, forexample, by multiple colors. Alternatively, the pattern may be focusedon a particular portion or portions of a genome, such as a portion orportions potentially containing a deletion or breakpoint that isdiagnostically or prognostically important for one or more tumors, or onthose portions of chromosomes having significance for prenataldiagnosis.

The staining patterns may be adjusted for the analysis method employed,for example, either a human observer or automated equipment, such as,flow cytometers or computer assisted microscopy. The patterns may bechosen to be appropriate for analysis of condensed chromosomes ordispersed chromosomal material.

The invention further provides for automated means of detecting andanalyzing chromosomal abnormalities as indicated by the stainingpatterns produced according to this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, B and C and FIGS. 2A and 2B illustrate the hybridization of achromosome-specific 21 library to human metaphase spread wherein theinserts were cloned in Lambda phage Charon 21A. The hybridizationcapacity of the high copy repetitive sequences in the library wasreduced by the addition of unlabeled genomic DNA to the hyridizationmixture. The probe was labeled with biotin, which was detected withgreen FITC-avidin (fluorescein isothiocyanate avidin). All of the DNA inthe chromosomes was stained with the blue fluorescent dye DAPI(4,6-diamidino-2-phenylindole).

FIG. 1A is a binary image of the DAPI stain in the human metaphasespread obtained by using a TV camera attached to a fluorescencemicroscope. Filters appropriate for DAPI visualization were used.Computer processing of the image shows all portions above a chosenthreshold intensity as white, and the rest as black.

FIG. 1B is a binary image of the FITC staining of the same humanmetaphase spread as in FIG. 1A. The image was processed as in FIG. 1Abut the filter was changed in the microscope such that the FITC attachedto the probe is visible rather than the DAPI.

FIG. 1C is a binary image of the chromosome 21s alone, nonspecificallystained objects (which are smaller) having been removed by standardimage processing techniques on the binary image of FIG. 1B.

FIG. 2A is a black-and-white photograph of the DAPI stain in a humanmetaphase spread which was prepared and hybridized contemporaneouslywith the spread shown in the computer generated binary images of FIGS.1A, B and C.

FIG. 2B is a black-and-white photograph of the fluorescein attached tothe DNA probe in the same human metaphase spread as shown in FIG. 2A. Itwas obtained by changing the filters in the fluorescence microscope toexcite fluorescein rather than DAPI. The photograph is comparable to thebinary image of FIG. 1B.

FIG. 3 is a photograph of a human metaphase spread prepared andhybridized contemporaneously with the spreads shown in FIGS. 1A, B and Cand 2A and B. The procedures used were the same except that PI(propidium iodide) instead of DAPI, was used to stain all thechromosomes. Both PI and fluorescein stains can be viewed with the samemicroscope filters. Color film was used such that the propidium iodidecounterstain appears red and the fluorescein of the probe appears yellowon the color film.

FIG. 4A shows the hybridization of the chromosome 4-specific library inBluescribe plasmids (the library pBS-4) to a human metaphase spreadwherein no unlabeled human genomic DNA was used, and wherein thehybridization mixture was applied immediately after denaturation. Bothcopies of chromosome 4 are seen as slightly brighter than the otherchromosomes. The small arrows indicate regions that are unstained withthe probe. As in FIG. 3 and as in the rest of the Figures below, PI isthe counterstain and fluorescein is used to label the probe.

FIG. 4B shows the hybridization of pBS-4 to a human metaphase spreadwherein unlabeled human genomic DNA was used during the hybridization(Q=2 of genomic DNA; the meaning of Q is explained infra). Quantitativeimage analysis shows that the intensity per unit length of the chomosome4s is about 20× that of the other chromosomes. The chromosome 4s areyellow; the other chromosomes are red due to the propidium iodidecounterstain. Two layers of avidin-fluorescein isothiocyanate have beenused to make the target chromosomes sufficiently bright to be measuredaccurately. However, the number 4 chromosomes can be recognized easilyafter a single layer is applied.

FIG. 4C shows the same spread as in FIG. 4B but through a filter thatpasses only the fluorescein isothiocyanate fluorescence.

FIG. 4D shows the detection of a radiation-induced translocation(arrows) involving chromosome 4s in a human metaphase spread whereinpBS-4 specific libraries are used. The contrast ratio is about 5×.

FIG. 4E shows that normal and two derivative chromosomes resulting froma translocation between chromosome 4 and 11 (in cell line RS4;11) can bedetected by the compositions and methods of this invention in interphasenuclei. They appear as three distinct domains.

FIG. 4F shows the hybridization of the chromosome 21-specific library inBluescribe plasmids (the library pBS-21) to a metaphase spread of atrisomy 21 cell line. A small amount of hybridization is visible nearthe centromeres of the other acrocentric chromosomes.

FIG. 4G shows the same hybridization as in FIG. 4F but with interphasenuclei. Clearly shown are the three chromosome 21 domains.

FIG. 4H shows the hybridization with a pool of 120 single copy probesfrom chromosome 4 to a human metaphase spread. The number 4 chromosomesare indicated by arrows.

FIG. 5 shows the hybridization of a yeast artificial chromosome (YAC)clone containing a 580 kb insert of human DNA to a human metaphasespread. A yellow fluorescein band on each of the chromosome 12s (at12q21.1) is visible against the propidium iodide counterstain.

FIG. 6 shows the hybridization of DNA from a human/hamster hybrid cellcontaining one copy of human chromosome 19 to a human metaphase spread.A little to the right of the photograph's center are the two chromosome19s which are brighter than the other chromosomes in the spread.

FIG. 7 illustrates a representative method of using the polymerase chainreaction (PCR) to produce probes of this invention which are reduced inrepetitive sequences.

DETAILED DESCRIPTION OF THE INVENTION

This invention concerns the use of nucleic acid probes to stain targetedchromosomal material in patterns which can extend along one or morewhole chromosomes, and/or along one or more regions on one or morechromosomes, including patterns which extend over an entire genome. Thestaining reagents of this invention facilitate the microscopic and/orflow cytometric identification of normal and aberrant chromosomes andprovide for the characterization of the genetic nature of particularabnormalities. The term "chromosome-specific" is herein defined toencompass the terms "target specific" and "region specific", that is,when the staining composition is directed to one chromosome, it ischromosome-specific, but it is also chromosome-specific when it isdirected, for example, to multiple regions on multiple chromosomes, orto a region of only one chromosome, or to regions across the entiregenome. The term chromosome-specific originated from the use ofrecombinant DNA libraries made by cloning DNA from a single normalchromosome type as the source material for the initial probes of thisinvention. Libraries made from DNA from regions of one or morechromosomes are sources of DNA for probes for that region or thoseregions of the genome. The probes produced from such source material areregion-specific probes but are also encompassed within the broaderphrase "chromosome-specific" probes. The term "target specific" isinterchangeably used herein with the term "chromosome-specific".

The word "specific" as commonly used in the art has two somewhatdifferent meanings. The practice is followed herein. "Specific" mayrefer to the origin of a nucleic acid sequence or to the pattern withwhich it will hybridize to a genome as part of a staining reagent. Forexample, isolation and cloning of DNA from a specified chromosomeresults in a "chromosome-specific library". E.g., Van Dilla et al.,"Human Chromosome-Specific DNA Libraries: Construction andAvailability," Biotechnology, 4:537 (1986).! However, such a librarycontains sequences that are shared with other chromosomes. Such sharedsequences are not chromosome-specific to the chromosome from which theywere derived in their hybridization properties since they will bind tomore than the chromosome of origin. A sequence is "chromosome-specific"if it binds only to the desired portion of a genome. Such sequencesinclude single-copy sequences contained in the target or repetitivesequences, in which the copies are contained predominantly in thetarget.

"Chromosome-specific" in modifying "staining reagent" refers to theoverall hybridization pattern of the nucleic acid sequences thatcomprise the reagent. A staining reagent is chromosome-specific ifuseful contrast between the target and non-target chromosomal materialis achieved (that is, that the target can be adequately visualized).

A probe is herein defined to be a collection of nucleic acid fragmentswhose hybridization to the target can be detected. The probe is labeledas described below so that its binding to the target can be visualized.The probe is produced from some source of nucleic acid sequences, forexample, a collection of clones or a collection of polymerase chainreaction (PCR) products. The source nucleic acid may then be processedin some way, for example, by removal of repetitive sequences or blockingthem with unlabeled nucleic acid with complementary sequence, so thathybridization with the resulting probe produces staining of sufficientcontrast on the target. Thus, the word probe may be used herein to refernot only to the detectable nucleic acid, but also to the detectablenucleic acid in the form in which it is applied to the target, forexample, with the blocking nucleic acid, etc. The blocking nucleic acidmay also be mentioned separately. What "probe" refers to specificallyshould be clear from the context in which the word is used.

The term "labeled" is herein used to indicate that there is some methodto visualize the bound probe, whether or not the probe directly carriessome modified constituent. Section III infra describes various means ofdirectly labeling the probe and other labeling means by which the boundprobe can be detected.

The terms "staining" or "painting" are herein defined to meanhybridizing a probe of this invention to a genome or segment thereof,such that the probe reliably binds to the targeted chromosomal materialtherein and the bound probe is capable of being visualized. The terms"staining" or "painting" are used interchangeably. The patternsresulting from "staining" or "painting" are useful for cytogeneticanalysis, more particularly, molecular cytogenetic analysis. Thestaining patterns facilitate the microscopic and/or flow cytometricidentification of normal and abnormal chromosomes and thecharacterization of the genetic nature of particular abnormalities.Section III infra describes methods of rendering the probe visible.Since multiple compatible methods of probe visualization are available,the binding patterns of different components of the probe can bedistinguished--for example, by color. Thus, this invention is capable ofproducing any desired staining pattern on the chromosomes visualizedwith one or more colors (a multi-color staining pattern) and/or otherindicator methods. The term "staining" as defined herein does notinclude the concept of staining chromosomes with chemicals as inconventional karotyping methods although such conventional stains may beused in conjunction with the probes of this invention to allowvisualization of those parts of the genome where the probe does notbind. The use of DAPI and propidium iodide for such a purpose isillustrated in the figures.

The phrase "high complexity" is defined herein to mean that the probe,thereby modified contains on the order of 50,000 (50 kb) or greater, upto many millions or several billions, of bases of nucleic acid sequenceswhich are not repeated in the probe. For example, representative highcomplexity nucleic acid probes of this invention can have a complexitygreater than 50 kb, greater than 100,000 bases (100 kb), greater than200,000 (200 kb), greater than 500,000 bases (500 kb), greater than onemillion bases (1 Mb), greater than 2 Mb, greater than 10 Mb, greaterthan 100 Mb, greater than 500 Mb, greater than 1 billion bases and stillfurther greater than several billion bases.

The term "complexity" is defined herein according to the standard fornucleic acid complexity as established by Britten et al., Methods ofEnzymol., 29:363 (1974). See also Cantor and Schimmel, BiophysicalChemistry: Part III: The Behavior of Biological Macromolecules, at1228-1230 (Freeman and Co. 1980) for further explanation andexemplification of nucleic acid complexity.

The complexity preferred for a probe composition of this invention isdependent upon the application for which it is designed. In general, thelarger the target area, the more complex is the probe. It is anticipatedthat the complexity of a probe needed to produce a desired pattern oflandmarks on a chromosome will decrease as hybridization sensitivityincreases, as progress is made in hybridization technology. As thesensitivity increases, the reliability of the signal from smaller targetsites will increase. Therefore, whereas from about a 40 kb to about a100 kb target sequence may be presently necessary to provide a reliable,easily detectable signal, smaller target sequences should providereliable signals in the future. Therefore, as hybridization sensitivityincreases, a probe of a certain complexity, for example, 100 kb, shouldenable the user to detect considerably more loci in a genome than arepresently reliably detected; thus, more information will be obtainedwith a probe of the same complexity. The term "complexity" thereforerefers to the complexity of the total probe no matter how many visuallydistinct loci are to be detected, that is, regardless of thedistribution of the target sites over the genome.

As indicated above, with current hybridization techniques it is possibleto obtain a reliable, easily detectable signal with a probe of about 40kb to about 100 kb (e.g. the probe insert capacity of one or a fewcosmids) targeted to a compact point in the genome. Thus, for example, acomplexity in the range of approximately 100 kb now permitshybridization to both sides of a tumor-specific translocation. Theportion of the probe targeted to one side of the breakpoint can belabeled differently from that targeted to the other side of thebreakpoint so that the two sides can be differentiated with differentcolors, for example. Proportionately increasing the complexity of theprobe permits analysis of multiple compact regions of the genomesimultaneously. The conventional banding patterns produced by chemicalstains may be replaced according to this invention with a series ofprobe-based, color coded (for example), reference points along eachchromosome or significant regions thereof.

Uniform staining of an extended contiguous region of a genome, forexample, a whole chromosome, requires a probe complexity proportional tobut substantially less than, the complexity of the target region. Thecomplexity required is only that necessary to provide a reliable,substantially uniform signal on the target. Section V.B, infra,demonstrates that fluorescent staining of human chromosome 21, whichcontains about 50 megabases (Mb) of DNA, is sufficient with a probecomplexity of about 1 Mb. FIG. 4H illustrates hybridization of about 400kb of probe to human chromosome 4, which contains about 200 Mb of DNA.In that case, gaps between the hybridization of individual elements ofthe probe are visible. FIGS. 4B and 4F demonstrate the results achievedwith probes made up of entire libraries for chromosomes 4 and 21,respectively. The chromosomes are stained much more densely as shown inFIGS. 4B and 4F than with the lower complexity probe comprisingsingle-copy nucleic acid sequences used to produce the pattern of FIG.4H.

Increasing the complexity beyond the minimum required for adequatestaining is not detrimental as long as the total nucleic acidconcentration in the probe remains below the point where hybridizationis impaired. The decrease in concentration of a portion of a sequence inthe probe is compensated for by the increase in the number of targetsites. In fact, when using double-stranded probes, it is preferred tomaintain a relatively low concentration of each portion of sequence toinhibit reassociation before said portion of sequence can find a bindingsite in the target.

The staining patterns of this invention comprise one or more "bands".The term "band" is herein defined as a reference point in a genome whichcomprises a target nucleic acid sequence bound to a probe component,which duplex is detectable by some indicator means, and which at itsnarrowest dimension provides for a reliable signal under the conditionsand protocols of the hybridization and the instrumentation, among othervariables, used. A band can extend from the narrow dimension of asequence providing a reliable signal to a whole chromosome to multipleregions on a number of chromosomes.

The probe-produced bands of this invention are to be distinguished frombands produced by chemical staining as indicated above in theBackground. The probe-produced bands of this invention are based uponnucleic acid sequence whereas the bands produced by chemical stainingdepend on natural characteristics of the chromosomes, but not the actualnucleic acid sequence. Further, the banding patterns produced bychemical staining are only interpretable in terms of metaphasechromosomes whereas the probe-produced bands of this invention areuseful both for metaphase and interphase chromosomes.

One method of forming the probes of the present invention is to poolmany different low complexity probes. Such a probe would then comprise a"heterogeneous mixture" of individual cloned sequences. The number ofclones required depends on the extent of the target area and thecapacity of the cloning vector. If the target is made up of severaldiscrete, compact loci, that is, single spots at the limit ofmicroscopic resolution, then about 40 kb, more preferably 100 kb, foreach spot gives a reliable signal given current techniques. The portionof the probe for each spot may be made up from, for example, a singleinsert from a yeast artificial chromosome (YAC), from several cosmidseach containing 35-40 kb or probe sequence, or from about 25 plasmidseach with 4 kb of sequence.

Representative heterogeneous mixtures of clones exemplified hereininclude phage (FIGS. 1, 2 and 3), and plasmids (FIG. 4). Yeastartificial chromosomes (YACS) (FIG. 5), and a single human chromosome inan inter-species hybrid cell (FIG. 6) are examples of high complexityprobes for single loci and an entire chromosome that can be propagatedas a single clone.

A base sequence at any point in the genome can be classified as either"single-copy" or "repetitive". For practical purposes the sequence needsto be long enough so that a complementary probe sequence can form astable hybrid with the target sequence under the hybridizationconditions being used. Such a length is typically in the range ofseveral tens to hundreds of nucleotides.

A "single-copy sequence" is that wherein only one copy of the targetnucleic acid sequence is present in the haploid genome. "Single-copysequences" are also known in the art as "unique sequences". A"repetitive sequence" is that wherein there are more than one copy ofthe same target nucleic acid sequence in the genome. Each copy of arepetitive sequence need not be identical to all the others. Theimportant feature is that the sequence be sufficiently similar to theother members of the family of repetitive sequences such that under thehybridization conditions being used, the same fragment of probe nucleicacid is capable of forming stable hybrids with each copy. A "sharedrepetitive sequence" is a sequence with some copies in the target regionof the genome, and some elsewhere.

When the adjectives "single-copy", "repetitive", "shared repetitive",among other such modifiers, are used to describe sequences in the probe,they refer to the type of sequence in the target to which the probesequence will bind. Thus, "a repetitive probe" is one that binds to arepetitive sequence in the target; and "a single-copy probe" binds to asingle-copy target sequence.

Repetitive sequences occur in multiple copies in the haploid genome. Thenumber of copies can range from two to hundreds of thousands, whereinthe Alu family of repetitive DNA are exemplary of the latter numerousvariety. The copies of a repeat may be clustered or interspersedthroughout the genome. Repeats may be clustered in one or more locationsin the genome, for example, repetitive sequences occurring near thecentromeres of each chromosome, and variable number tandem repeats(VNTRs) Nakamura et al, Science, 235: 1616 (1987)!; or the repeats maybe distributed over a single chromosome for example, repeats found onlyon the X chromosome as described by Bardoni et al., Cytogenet. CellGenet., 46: 575 (1987)!; or the repeats may be distributed over all thechromosomes, for example, the Alu family of repetitive sequences.

Herein, the terms repetitive sequences, repeated sequences and repeatsare used interchangeably.

Shared repetitive sequences can be clustered or interspersed. Clusteredrepetitive sequences include tandem repeats which are so named becausethey are contiguous on the DNA molecule which forms the backbone of achromosome. Clustered repeats are associated with well-defined regionsof one or more chromosomes, e.g., the centromeric region. If one or moreclustered repeats form a sizable fraction of a chromosome, and areshared with one or more non-target regions of the genome and areconsequently removed from the heterogeneous mixture of fragmentsemployed in the invention or the hybridization capacity thereof isdisabled, perfect uniformity of staining of the target region may not bepossible. That situation is comprehended by the use of the term"substantially uniform" in reference to the binding of the heterogeneousmixture of labeled nucleic acid fragments to the target.

Chromosome-specific staining of the current invention is accomplished byusing nucleic acid fragments that hybridize to sequences specific to thetarget. These sequences may be either single-copy or repetitive, whereinthe copies of the repeat occur predominantly in the target area. FIG. 4Hand the results of the work detailed in section V infra indicate thatprobes can be made of single-copy sequences. However, in probes such asthat of FIG. 4B, low-copy chromosome-specific repeats Nakamura et al.,and Bardoni et al., supra! may contribute to the hybridization as well.

If nucleic acid fragments complementary to non-target regions of thegenome are included in the probe, for example, shared repetitivesequences or non-specific sequences, their hybridization capacity needsto be sufficiently disabled or their prevalence sufficiently reduced, sothat adequate staining contrast can be obtained. Section V and FIG. 4Hshow examples of hybridization with probes that contain pools of clonesin which each clone has been individually selected so that it hybridizesto single-copy sequences or very low copy repetitive sequences. Theremaining figures illustrate use of probes that contain fragments thatcould have hybridized to high-copy repetitive sequences, but which havehad the hybridization capacity of such sequences disabled.

The nucleic acid probes of this invention need not be absolutelyspecific for the targeted portion of the genome. They are intended toproduce "staining contrast". "Contrast" is quantified by the ratio ofthe stain intensity of the target region of the genome to that of theother portions of the genome. For example, a DNA library produced bycloning a particular chromosome, such as those listed in Table I, can beused as a probe capable of staining the entire chromosome. The librarycontains sequences found only on that chromosome, and sequences sharedwith other chromosomes. In a simplified (approximately true to life)model of the human genome, about half of the chromosomal DNA falls intoeach class. If hybridization with the whole library were capable ofsaturating all of the binding sites, the target chromosome would betwice as bright (contrast ratio of 2) as the others since it wouldcontain signal from the specific and shared sequences in the probe,whereas the other chromosome would only have signal from the sharedsequences. Thus, only a modest decrease in hybridization of the sharedsequences in the probe would substantially enhance the contrast.Contaminating sequences which only hybridize to non-targeted sequences,for example, impurities in a library, can be tolerated in the probe tothe extent that said sequences do not reduce the staining contrast belowuseful levels.

In reality all of the target sites may not be saturated during thehybridization, and many other mechanisms contribute to producingstaining contrast, but this model illustrates one general considerationin using probes targeted at a large portion of a genome.

The required contrast depends on the application for which the probe isdesigned. When visualizing chromosomes and nuclei, etc.,microscopically, a contrast ratio of two or greater is often sufficientfor identifying whole chromosomes. In FIGS. 4D-F, the contrast ratio is3-5. The smaller the individual segments of the target region, thegreater the contrast needs to be to permit reliable recognition of thetarget relative to the fluctuations in staining of the non-targetedregions. When quantifying the amount of target region present in a cellnucleus by fluorescence intensity measurements using flow cytometry orquantitative microscopy, the required contrast ratio is on the order of1/T or greater on average for the genome, where T is the fraction of thegenome contained in the targeted region. When the contrast ratio isequal to 1/T, half of the total fluorescence intensity comes from thetarget region and half from the rest of the genome. For example, whenusing a high complexity probe for chromosome 1, which comprises about10% of the genome, the required contrast ratio is on the order of 10,that is, for the chromosome 1 fluorescence intensity to equal that ofthe rest of the genome.

Background staining by the probe, that is, to the non-target region ofthe genome, may not be uniform. FIG. 4F shows that a chromosome 21specific probe contains probe fragments that hybridize weakly to compactregions near the centromeres of other acrocentric human chromosomes.This degree of non-specificity does not inhibit its use in theillustrated applications. For other applications, removal of or furtherdisabling the hybridization capacity of the probe fragments that bind tothese sequences may be necessary.

For other applications, repetitive sequences that bind to centromeres,for example, alpha-satellite sequences, and/or telomeres can be part ofthe chromosome-specific staining reagents wherein the target includessome or all of the centromeres and/or telomeres in a genome along withperhaps other chromosomal regions. Exemplary of such an applicationwould be that wherein the staining reagent is designed to detect randomstructural aberrations caused by clastogenic agents that result indicentric chromosomes and other structural abnormalities, such astranslocations. Addition of sequences which bind to all centromeres in agenome, for example to the probe used to create the staining pattern ofFIG. 4D, would allow more reliable distinguishing between dicentrics andtranslocations.

Application of staining reagents of this invention to a genome resultsin a substantially uniform distribution of probe hybridized to thetargeted regions of a genome. The distribution of bound probe is deemed"substantially uniform" if the targeted regions of the genome can bevisualized with useful contrast. For example, a target is substantiallyuniformly stained in the case wherein it is a series of visuallyseparated loci if most of the loci are visible in most of the cells.

"Substantial proportions" in reference to the base sequences of nucleicacid fragments that are complementary to chromosomal DNA means that thecomplementarity is extensive enough so that the fragments form stablehybrids with the chromosomal DNA under the hybridization conditionsused. In particular, the term comprehends the situation where thenucleic acid fragments of the heterogeneous mixture possess some regionsof sequence that are not perfectly complementary to target chromosomalmaterial. The stringency can be adjusted to control the precision of thecomplementarity required for hybridization.

The phrase "metaphase chromosomes" is herein defined to mean not onlychromosomes condensed in the metaphase stage of mitosis but includes anycondensed chromosomes, for example, those condensed by prematurechromosome condensation.

To disable the hybridization capacity of a nucleic acid sequence isherein sometimes abbreviated as "disabling the nucleic acid sequence".

The following sections provide examples of making and using the stainingcompositions of this invention and are for purposes of illustration onlyand not meant to limit the invention in any way. The followingabbreviations are used.

Abbreviations

BN--bicarbonate buffer with NP-40

DAPI--4,6-diamidino-2-phenylindole

DCS--as in fluorescein-avidin DCS (a commercially available cell sortergrade of fluorescein Avidin D)

AAF--N-acetoxy-N-2-acetyl-aminofluorene

EDTA--ethylenediaminetetraacetate

FACS--fluorescence-activated cell sorting

FITC--fluorescein isothiocyanate

IB--isolation buffer

NP-40-non-ionic detergent commercially available from Sigma as NonidetP-40 (St. Louis, Mo.)

PBS--phosphate-buffered saline

PI--propidium iodide

PMSF--phenylmethylsulfonyl fluoride

PN--mixture of 0.1M NaH₂ PO₄ and 0.1M buffer Na₂ HPO₄, pH 8; 0.1% NP-40

PNM--Pn buffer plus 5% nonfat dry milk (centrifuged); buffer 0.02% Naazide

SDS--sodium dodecyl sulfate

SSC--0.15M NaCl/0.015M Na citrate, pH 7

VNTR--variable number tandem repeat

I. Methods of Preparing Chromosome-Specific Staining Reagents

I.A. Isolation of Chromosome-Specific DNA and Formation of DNA FragmentLibraries

The first step in a preferred method of making the compositions of theinvention is isolating chromosome-specific DNA (which term includestarget-specific and/or region-specific DNA, as indicated above, whereinspecific refers to the origin of the DNA). This step includes firstisolating a sufficient quantity of the particular chromosome type orchromosomal subregion to which the staining composition is directed,then extracting the DNA from the isolated chromosome(s) or chromosomalsubregion(s). Here "sufficient quantity" means sufficient for carryingout subsequent steps of the method. Preferably, the extracted DNA isused to create a library of DNA inserts by cloning using standardgenetic engineering techniques.

Preferred cloning vectors include, but are not limited to, yeastartificial chromosomes (YACS), plasmids, bacteriophages and cosmids.Preferred plasmids are Bluescribe plasmids; preferred bacteriophages arelambda insertion vectors, more preferably Charon 4A, Charon 21A, Charon35, Charon 40 and GEM11; and preferred cosmids include Lawrist 4,Lawrist 5 and sCos1.

As indicated above, the DNA can be isolated from any source.Chromosome-specific staining reagents can be made from both plant andanimal DNA according to the methods of this invention. Important sourcesof animal DNA are mammals, particularly primates or rodents whereinprimate sources are more particularly human and monkey, and rodentsources are more particularly rats or mice, and more particularly mice.

1. Isolating DNA from an Entire Chromosome. A preferred means forisolating particular whole chromosomes (specific chromosome types) is bydirect flow sorting fluorescence-activated cell sorting (FACS)! ofmetaphase chromosomes with or without the use of interspecific hybridcell systems. For some species, every chromosome can be isolated bycurrently available sorting techniques. Most, but not all, humanchromosomes are currently isolatable by flow sorting from human cells,Carrano et al., "Measurement and Purification of Human Chromosomes byFlow Cytometry and Sorting," Proc. Natl. Acad. Sci., Vol. 76, pgs.1382-1384 (1979). Thus, for isolation of some human chromosomes, use ofthe human/rodent hybrid cell system may be necessary, see Kao, "SomaticCell Genetics and Gene Mapping," International Review of Cytology. Vol.85, pgs. 109-146 (1983), for a review, and Gusella et al., "Isolationand Localization of DNA Segments from Specific Human Chromosomes," Proc.Natl. Acad. Sci., Vol. 77, pgs. 2829-2833 (1980). Chromosome sorting canbe done by commercially available fluorescence-activated sortingmachines, e.g., Becton Dickinson FACS-II, Coulter Epics V sorter, orspecial purpose sorters optimized for chromosome sorting or likeinstrument.

DNA is extracted from the isolated chromosomes by standard techniques,e.g., Marmur, "A Procedure for the Isolation of Deoxyribonucleic Acidfrom Micro-Organisms," J. Mol. Biol., Vol. 3, pgs. 208-218 (1961); orManiatis et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory, 1982) pgs. 280-281. These references are incorporatedby reference for their descriptions of DNA isolation techniques.

Generation of insert libraries from the isolated chromosome-specific DNAis carried out using standard genetic engineering techniques, e.g.,Davies et al., "Cloning of a Representative Genomic Library of the HumanX Chromosome After Sorting by Flow Cytometry," Nature, Vol. 293, pgs.374-376 (1981); Krumlauf et al., "Construction and Characterization ofGenomic Libraries from Specific Human Chromosomes," Proc. Natl. Acad.Sci., Vol. 79, pgs. 2971-2975 (1982); Lawn et al., "The Isolation andCharacterization of Linked Delta-and-Beta-Globin Genes from a ClonedLibrary of Human DNA." Cell, Vol. 15, pgs. 1157-1174 (1978); andManiatis et al., "Molecular Cloning: A Laboratory Manual," (Cold SpringsHarbor Laboratory, 1982), pgs. 256-308; Van Dilla et al., id; Fuscoe,Gene, 52:291 (1987); and Fuscoe et al., Cytogenet. Cell Genet., 43:79(1986). Said references are herein incorporated by reference.

Recombinant DNA libraries for each of the human chromosomes have beenconstructed by the National Laboratory Gene Library Project and areavailable from the American Type Culture Collection. Van Dilla et al.,Biotechnology, 4:537 (1986).! Small insert-containing libraries wereconstructed by complete digestion of flow sorted human chromosomegenomic DNA with HindIII or EcoRI and cloning into the Lambda insertionvector Charon 21A. The vector is capable of accepting human inserts ofup to 9.1 kb in size. Thus, HindIII (or EcoRI) restriction fragmentsgreater than 9.1 kb will not be recovered from these libraries. Theobserved average insert size in these libraries is approximately 4 kb. Arepresentative list of the HindIII chromosome-specific libraries withtheir ATCC accession numbers are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        HUMAN CHROMOSOME - SPECIFIC GENOMIC LIBRARIES                                 IN CHARON 21A VECTOR                                                          CHROMOSOME      ATCC #   LIBRARY                                              ______________________________________                                         1              57753    LL01NS01                                              1              57754    LL01NS02                                              2              57744    LL02NS01                                              3              57751    LL03NS01                                              4              57700    LL04NS01                                              4              57745    LL04NS02                                              5              57746    LL05NS01                                              6              57701    LL06NS01                                              7              57755    LL07NS01                                              8              57702    LL08NS02                                              9              57703    LL09NS01                                             10              57736    LL10NS01                                             11              57704    LL11NS01                                             12              57756    LL12NS01                                             13              57705    LL13NS01                                             13              57757    LL13NS02                                             14              57706    LL14NS01                                             14/15           57707    LL99NS01                                             15              57737    LL15NS01                                             16              57758    LL16NS03                                             17              57759    LL17NS02                                             18              57710    LL18NS01                                             19              57711    LL19NS01                                             20              57712    LL20NS01                                             21              57713    LL21NS02                                             22              57714    LL22NS01                                             X               57747    LL0XNS01                                             Y               57715    LL0YNS01                                             ______________________________________                                    

Alternatively, the extracted DNA from a sorted chromosome type can beamplified by the polymerase chain reaction (PCR) rather than cloning theextracted DNA in a vector or propagating it in a cell line. Appropriatetails are added to the extracted DNA in preparation for PCR. Referencesfor such PCR procedures are set out in Section I.B infra.

Other possible methods of isolating the desired sequences from hybridcells include those of Schmeckpeper et al., "Partial Purification andCharacterization of DNA from Human X Chromosome," Proc. Natl. Acad.Sci., Vol. 76, pgs. 6525-6528 (1979); or Olsen et al., supra (inBackground). Accordingly, these references are incorporated byreference.

2. Isolating DNA from a Portion of a Chromosome. Among the methods thatcan be used for isolating region-specific chromosomal DNA include theselection of an appropriate chromosomal region from DNA that haspreviously been mapped, for example, from a library of mapped cosmids;the sorting of derivative chromosomes, for example, by FACS; themicrodissection of selected chromosomal material; subtractivehybridization; identification of an appropriate hybrid cell containing adesired chromosomal fragment, extracting and amplifying the DNA, andselecting the desired amplified DNA; and the selection of appropriatechromosomal material from radiation hybrids. The standard geneticengineering techniques outlined above in subsection I.A.1 are used insuch procedures well-known to those in the art. Amplification of theregion-specific DNA can be performed by cloning in an appropriatevector, propagating in an appropriate cell line, and/or by the use ofPCR (see I.B infra).

A preferred method of isolating chromosomal region-specific DNA is touse mapped short DNA sequences to probe a library of longer DNAsequences, wherein the latter library has usually been cloned in adifferent vector. For example, a probe cloned in a plasmid can be usedto probe a cosmid or yeast artificial chromosome (YAC) library. By usingan initial seed probe, overlapping clones in the larger insert librarycan be found (a process called "walking"), and a higher complexity probecan be produced for reliable staining of the chromosomal regionsurrounding the seed probe. Ultimately, when an entire genome for aspecies has been mapped (for example, by the Human Genome Project forthe human species), ordered clones for the entire genome of the specieswill be available. One can then easily select the appropriate clones toform a probe of the desired specificity.

Another method of isolating DNA from a chromosomal region or regions (oralso a whole chromosome) is to propagate such a chromosomal region orregions in an appropriate cell line (for example, a hybrid cell linesuch as a human/hamster hybrid cell), extract the DNA from the cell lineand clone it in an appropriate vector and select clones containing humanDNA to form a library. When a hybrid cell is used, the chromosomes inthe hybrid cell containing the human chromosomal material may beseparated by flow sorting (FACS) prior to cloning to increase thefrequency of human clones in the library. Still further, total DNA fromthe hybrid cell can be isolated and labeled without further cloning andused as a probe, as exemplified in FIG. 6.

3. Single-Stranded Probes. In some cases, it is preferable that thenucleic acid fragments of the heterogeneous mixture consist ofsingle-stranded RNA or DNA. Under some conditions, the bindingefficiency of single-stranded nucleic acid probes has been found to behigher during in situ hybridization, e.g., Cox et al., "Detection ofmRNAs in Sea Urchin Embryos by In Situ Hybridization Using AsymmetricRNA Probes," Developmental Biology, Vol. 101, pgs. 485-502 (1984).

Standard methods are used to generate RNA fragments from isolated DNAfragments. For example, a method developed by Green et al., described inCell, Vol. 32, pgs. 681-694 (1983), is commercialy available fromPromega Biotec (Madison, Wis.) under the tradename "Riboprobe." Othertranscription kits suitable for use with the present invention areavailable from United States Biochemical Corporation (Cleveland, Ohio)under the tradename "Genescribe." Single-stranded DNA probes can beproduced with the single-stranded bacteriophage M13, also available inkit form, e.g. Bethesda Research Laboratories (Gaithersburg, Md.). Thehybridizations illustrated in FIG. 4 were performed with the librariesof Table 1 subcloned into the Bluescribe plasmid vector (Stratagene, LaJolla, Calif.). The Bluescribe plasmid contains RNA promoters whichpermit production of single-stranded probes.

Co-pending, commonly owned U.S. patent application Ser. No. 934,188(filed Nov. 24, 1986), entitled "Method of Preparing and Applying SingleStranded DNA Probes to Double Stranded Target DNAs," provides methodsfor preparing and applying non-self-complementary single-strandednucleic acid probes that improve signal-to-noise ratios attainable in insitu hybridization by reducing non-specific and mismatched binding ofthe probe. That application further provides for methods of denaturingdouble-stranded target nucleic acid which minimizes single-strandedregions available for hybridization that are non-complementary to probesequences. Said application is herein specifically incorporated byreference. Briefly, probe is constructed by treating DNA with arestriction enzyme and an exonuclease to form template/primers for a DNApolymerase. The digested strand is resynthesized in the presence oflabeled nucleoside triphosphate precursor, and the labeledsingle-stranded fragments are separated from the resynthesized fragmentsto form the probe. The target nucleic acid is treated with the samerestriction enzyme used to construct the probe, and is treated with anexonuclease before application of the probe.

I.B. PCR

Another method of producing probes of this invention includes the use ofthe polymerase chain reaction PCR!. For an explanation of the mechanicsof PCR, see Saiki et al., Science, 230:1350 (1985) and U.S. Pat. Nos.4,683,195, 4,683,202 (both issued Jul. 28, 1987) and 4,800,159 (issuedJan. 24, 1989).! Target-specific nucleic acid sequences, isolated asindicated above, can be amplified by PCR to produce target-specificsequences which are reduced in or free of repetitive sequences. The PCRprimers used for such a procedure are for the ends of the repetitivesequences, resulting in amplification of sequences flanked by therepeats.

FIG. 7 illustrates such a method of using PCR wherein the representativerepetitive sequence is Alu. If only short segments are amplified, it isprobable that such sequences are free of other repeats, thus providingDNA reduced in repetitive sequences.

One can further suppress production of repetitive sequences in such aPCR procedure by first hybridizing complementary sequences to saidrepetitive sequence wherein said complementary sequences have extendednon-complementary flanking ends or are terminated in nucleotides whichdo not permit extension by the polymerase. The non-complementary ends ofthe blocking sequences prevent the blocking sequences from acting as aPCR primer during the PCR process.

II. Removal of Repetitive Sequences and/or Disabling the HybridizationCapacity of Repetitive Sequences

Typically a probe of the current invention is produced in a number ofsteps including: obtaining source nucleic acid sequences that arecomplementary to the target region of the genome, labeling and otherwiseprocessing them so that they will hybridize efficiently to the targetand can be detected after they bind, and treating them to either disablethe hybridization capacity or remove a sufficient proportion of sharedrepetitive sequences, or both disable and remove such sequences. Theorder of these steps depends on the specific procedures employed.

The following methods can be used to remove shared repetitive sequencesand/or disable the hybridization capacity of such shared repetitivesequences. Such methods are representative and are expressedschematically in terms of procedures well known to those of ordinaryskill the art, and which can be modified and extended according toparameters and procedures well known to those in the art.

1. Single-copy probes. A single-copy probe consists of nucleic acidfragments that are complementary to single-copy sequences contained inthe target region of the genome. One method of constructing such a probeis to start with a DNA library produced by cloning the target region.Some of the clones in the library will contain DNA whose entire sequenceis single-copy; others will contain repetitive sequences; and stillothers will have portions of single-copy and repetitive sequences.Selection, on a clone by clone basis, and pooling of those clonescontaining only single-copy sequences will result in a probe that willhybridize specifically to the target region. The single-copy nature of aclone can ultimately be established by Southern hybridization usingstandard techniques. FIG. 4H shows hybridization with 120 clonesselected in this way from a chromosome 4 library.

Southern analysis is very time consuming and labor intensive. Therefore,less perfect but more efficient screening methods for obtainingcandidate single-copy clones are useful. In Section V.B, examples ofimproved methods are provided for screening individual phage and plasmidclones for the presence of repetitive DNA using hybridization withgenomic DNA. The screening of plasmid clones is more efficient, andapproximately 80% of selected clones contain only single-copy sequences;the remainder contain low-copy repeats. However, probes produced in thisway can produce adequate staining contrast, indicating that the low-copyrepetitive sequences can be tolerated in the probe (see subsection 3 ofthis section).

A disadvantage of clone by clone procedures is that a clone is discardedeven if only a portion of the sequence it contains is repetitive. Thelarger the length of the cloned nucleic acid, the greater the chancethat it will contain a repetitive sequence. Therefore, when nucleic acidis propagated in a vector that contains large inserts such as a cosmid,YAC, or in a cell line, such as hybrid cells, it may be advantageous tosubclone it in smaller pieces before the single-copy selection isperformed. The selection procedures just outlined above do notdiscriminate between shared and specific repetitive sequences; cloneswith detectable repetitive sequences of either type are not used in theprobe.

2. Individual testing of hybridization properties. The hybridizationspecificity of a piece of nucleic acid, for example, a clone, can betested by in situ hybridization. If under appropriate hybridizationconditions it binds to single-copy or repetitive sequences specific forthe desired target region, it can be included in the probe. Manysequences with specific hybridization characteristics are already known,such as chromosome-specific repetitive sequences Trask et al., supra,(1988) and references therein!, VNTRs, numerous mapped single copysequences. More are continuously being mapped. Such sequences can beincluded in a probe of this invention.

3. Bulk Procedures. In many genomes, such as the human genome, a majorportion of shared repetitive DNA is contained in a few families ofhighly repeated sequences such as Alu. A probe that is substantiallyfree of such high-copy repetitive sequences will produce useful stainingcontrast in many applications. Such a probe can be produced from somesource of nucleic acid sequences, for example, the libraries of Table I,with relatively simple bulk procedures. Therefore, such bulk proceduresare the preferred methods for such applications.

These methods primarily exploit the fact that the hybridization rate ofcomplementary nucleic acid strands increases as their concentrationincreases. Thus, if a heterogeneous mixture of nucleic acid fragments isdenatured and incubated under conditions that permit hybridization, thesequences present at high concentration will become double-stranded morerapidly than the others. The double-stranded nucleic acid can then beremoved and the remainder used as a probe. Alternatively, the partiallyhybridized mixture can be used as the probe, the double-strandedsequences being unable to bind to the target. The following are methodsrepresentative of bulk procedures that are useful for producing thetarget-specific staining of this invention.

3a. Self-reassociation of the probe. Double-stranded probe nucleic acidin the hybridization mixture is denatured and then incubated underhybridization conditions for a time sufficient for the high-copysequences in the probe to become substantially double-stranded. Thehybridization mixture is then applied to the sample. The remaininglabeled single-stranded copies of the highly repeated sequences bindthroughout the sample producing a weak, widely distributed signal. Thebinding of the multiplicity of low-copy sequences specific for thetarget region of the genome produce an easily distinguishable specificsignal.

Such a method is exemplified in Section VI.B (infra) withchromosome-specific libraries for chromosomes 4 and 21 (pBS4 and pBS21)as probes for those chromosomes. The hybridization mix, containing aprobe concentration in the range of 1-10 ng/ul was heated to denaturethe probe and incubated at 37° C. for 24 hours prior to application tothe sample.

3b. Use of blocking nucleic acid. Unlabeled nucleic acid sequences whichare complementary to those sequences in the probe whose hybridizationcapacity it is desired to inhibit are added to the hybridizationmixture. The probe and blocking nucleic acid are denatured, ifnecessary, and incubated under appropriate hybridization conditions. Thesequences to be blocked become double-stranded more rapidly than theothers, and therefore are unable to bind to the target when thehybridization mixture is applied to the target. In some cases, theblocking reaction occurs so quickly that the incubation period can bevery short, and adequate results can be obtained if the hybridizationmix is applied to the target immediately after denaturation. A blockingmethod is generally described by Sealy et al., "Removal of RepeatSequences form Hybridization Probes", Nucleic Acid Research, 13:1905(1985), which reference is incorporated by reference. Examples ofblocking nucleic acids include genomic DNA, a high-copy fraction ofgenomic DNA and particular sequences as outlined below (i-iii).

3b.i. Genomic DNA. Genomic DNA contains all of the nucleic acidsequences of the organism in proportion to their copy-number in thegenome. Thus, adding genomic DNA to the hybridization mixture increasesthe concentration of the high-copy repeat sequences more than low-copysequences, and therefore is more effective at blocking the former.However, the genomic DNA does contain copies of the sequences that arespecific to the target and so will also reduce the desiredchromosome-specific binding if too much is added. Guidelines todetermine how much genomic DNA to add (see

3.e. Concept of O, infra) and examples of using genomic blocking DNA areprovided below. The blocking effectiveness of genomic DNA can beenhanced under some conditions by adjusting the timing of its additionto the hybridization mix; examples of such timing adjustments areprovided with Protocol I and Protocol II hybridizations illustrated inFIGS. 4B through E (Protocol I) and FIG. 4F (Protocol II) and detailedin Section VI, infra.

3b.ii. Hiah-copy fraction of genomic DNA. The difficulty with use ofgenomic DNA is that it also blocks the hybridization of the low-copysequences, which are predominantly the sequences that give the desiredtarget staining. Thus, fractionating the genomic DNA to obtain only thehigh-copy sequences and using them for blocking overcomes thisdifficulty. Such fractionation can be done, for example, withhydroxyapatite as described below (3c.i).

3b.iii. Specified sequences. The blocking of a particular sequence inthe probe can be accomplished by adding many unlabeled copies of thatsequence. For example, Alu sequences in the probe can be blocked byadding cloned Alu DNA. Blocking DNA made from a mixture of a few clonescontaining the highest copy sequences in the human genome can be usedeffectively with chromosome-specific libraries for example, those ofTable I. Alternatively, unlabeled nucleic acid sequences from one ormore chromosome-specific libraries could be used to block a probecontaining labeled sequences from one or more other chromosome-specificlibraries. The shared sequences would be blocked whereas sequencesoccurring only on the target chromosome would be unaffected. FIG. 4Fshows that genomic DNA was not effective in completely blocking thehybridization of a sequence or sequences shared by human chromosome 21and the centromeric regions of the other human acrocentric chromosomes.When a clone or clones containing such a sequence or sequences is or areeventually isolated, unlabeled DNA produced therefrom could be added tothe genomic blocking DNA to improve the specificity of the staining.

3c. Removal of Sequences.

3c.i. Hydroxyapatite. Single- and double-stranded nucleic acids havedifferent binding characteristics to hydroxyapatite. Suchcharacteristics provide a basis commonly used for fractionating nucleicacids. Hydroxyapatite is commerically available (e.g. Bio-RadLaboratories, Richmond, Calif.). The fraction of genomic DNA containingsequences with a particular degree of repetition, from the highestcopy-number to single-copy, can be obtained by denaturing genomic DNA,allowing it to reassociate under appropriate conditions to a particularvalue of C_(o) t, followed by separation using hydroxyapatite. Thesingle- and double-stranded nucleic acid can also be discriminated byuse of S1 nuclease. Such techniques and the concept of C_(o) t areexplained in Britten et al., "Analysis of Repeating DNA Sequences byReassociation", in Methods in Enzymology, Vol. 29, pgs 363-418 (1974),which article is herein incorporated by reference.

The single-stranded nucleic acid fraction produced in 3a. or 3b. abovecan be separated by hydroxyapatite and used as a probe. Thus, thesequences that have been blocked (that become double-stranded) arephysically removed. The probe can then be stored until needed. The probecan then be used without additional blocking nucleic acid, or itsstaining contrast can perhaps be improved by additonal blocking.

3c.ii. Reaction with immobilized nucleic acid. Removal of particularsequences can also be accomplished by attaching single-stranded"absorbing" nucleic acid sequences to a solid support. Single-strandedsource nucleic acid is hybridized to the immobilized nucleic acid. Afterthe hybridization, the unbound sequences are collected and used as theprobe. For example, human genomic DNA can be used to absorb repetitivesequences from human probes. One such method is described by Brison etal., "General Method for Cloning Amplified DNA by Differential Screeningwith Genomic Probes," Molecular and Cellular Biology, Vol. 2, pgs.578-587 (1982). Accordingly, that reference is incorporated byreference. Briefly, minimally sheared human genomic DNA is bound todiazonium cellulose or a like support. The source DNA, appropriately cutinto fragments, is hybridized against the immobilized DNA to C_(o) tvalues in the range of about 1 to 100. The preferred stringency of thehybridization conditions may vary depending on the base composition ofthe DNA. Such a procedure could remove repetitive sequences fromchromosome-specific libraries, for example, those of Table I, to producea probe capable of staining a whole human chromosome.

3d. Blocking non-targeted sequences in the targeted genome. Blocking ofnon-targeted binding sites in the targeted genome by hybridization withunlabeled complementary sequences will prevent binding of labeledsequences in the probe that have the potential to bind to those sites.For example, hybridization with unlabeled genomic DNA will render thehigh-copy repetitive sequences in the target genome double-stranded.Labeled copies of such sequences in the probe will not be able to bindwhen the probe is subsequently applied.

In practice, several mechanisms combine to produce the stainingcontrast. For example, when blocking DNA is added to the probe as in 3babove, that which remains single-stranded when the probe is applied tothe target can bind to and block the target sequences. If the incubationof the probe with the blocking DNA is minimal, then the genomic DNAsimultaneously blocks the probe and competes with the probe for bindingsites in the target.

3e. Concept of O. As mentioned in section 3b.i above, it is necessary toadd the correct amount of genomic DNA to achieve the best compromisebetween inhibiting the hybridization capacity of high-copy repeats inthe probe and reducing the desired signal intensity by inhibition of thebinding of the target-specific sequences. The following discussionpertains to use of genomic blocking DNA with probes produced by cloningor otherwise replicating stretches of DNA from the target region of thegenome. Thus, the probe contains a representative sampling of thesingle-copy, chromosome-specific repetitive sequences, and sharedrepetitive sequences found in the target. Such a probe might range incomplexity from 100 kb of sequence derived from a small region of thegenome, for example several closely spaced cosmid clones; to manymillions of bases, for example a combination of multiple libraries fromTable I. The discussion below is illustrative and can be extended toother situations where different blocking nucleic acids are used. Thefollowing discussion of Q is designed only to give general guidelines asto how to proceed.

The addition of unlabeled genomic DNA to a hybridization mix containinglabeled probe sequences increases the concentration of all of thesequences, but increases the concentration of the shared sequences by alarger factor than the concentration of the target-specific sequencesbecause the shared sequences are found elsewhere in the genome, whereasthe target-specific sequences are not. Thus, the reassociation of theshared sequences is preferentially enhanced so that the hybridization ofthe labeled copies of the shared sequences to the target ispreferentially inhibited.

To quantity this concept, first consider one of the sequences, repeat orsingle-copy, that hybridize specifically to the ith chromosome in ahybridization mixture containing a mass m_(p) of probe DNA from the ithchromosome library of Table 1 (for example) and m_(b) of unlabeledgenomic DNA. The number of labeled copies of the sequence isproportional to m_(p). However, the number of unlabeled copies isproportional to f_(i) m_(b), where f_(i) is the fraction of genomic DNAcontained on the ith chromosome. Thus, the ratio of unlabeled to labeledcopies of each of the sequences specific for the target chromosome, isf_(i) m_(b) /m_(p), which is defined herein as Q. For normal humanchromosomes, 0.016≦f_(i) ≦0.08 Mendelsohn et al., Science, 179:1126(1973)!. For representative examples described in Section VI.B (infra),f₄ =0.066 and f₂₁ =0.016. For a probe targeted at a region comprised ofL base pairs, f_(i) =L/G where G is the number of base pairs in a genome(approximately 3×10⁹ bases for humans and other mammals). Thus, Q=(L/G)(m_(b) /m_(p)).

Now consider a shared sequence that is distributed more-or-lessuniformly over the genome, for example, Alu. The number of labeledcopies is proportional to m_(p), whereas the number of unlabeled copiesis proportional to m_(b). Thus, the ratio of unlabeled to labeled copiesis m_(b) /m_(p) =Q/f_(i). This is true for all uniformly distributedsequences, regardless of copy number. Thus adding genomic DNA increasesthe concentration of each specific sequence by the factor 1+Q, whereaseach uniformly distributed sequence is increased by the larger factor1+Q/f_(i). Thus, the reassociation rates of the shared sequences areincreased by a larger factor than those of the specific sequences by theaddition of genomic DNA.

It can be shown that roughly half of the beneficial effect of genomicDNA on relative reassociation rates is achieved when Q=1, and, by Q=5,there is essentially no more benefit to be gained by further increases.Thus, the protocol I hybridizations of Section VI.B infra keep Q≦5.

To illustrate the use of genomic blocking DNA, it is convenient toconsider a model of a genome wherein 50% of the DNA is comprised ofspecific sequences (both repetitive and single-copy) and the other 50%of the DNA is comprised of shared repetitive sequences that aredistributed uniformly over the genome. Thus, according to the model, ifthe target is L bases (that is, the probe contains fragmentsrepresenting L bases of the target area or areas of the genome),sequences containing L/2 bases will be specific to the target, and L/2will be shared with the entire genome.

Case I. The complexity of the probe is about 50 kb to about 100 kb. (Inthis case the complexity may be approximately equal to L since theprobability is that no repetitive sequences will typically occur withmore than a few copies in such a number of bases). Using a standardhybridization mixture (as exemplified in Section VI.B, infra), thetarget can be hybridized with about 2 ng of labeled probe DNA in 10 ulof hybridization mix, corresponding to approximately 1 pg/ul per kb ofspecific sequences (as used in Section VI.B, infra). Suppose thehybridization is to a slide containing 10⁴ cells (a typical number), andeach cell has about 6 pg of DNA, (typical for mammals). Then in thismodel calculation, there is 3 pg of shared repetitive sequences percell. Thus, for 10⁴ cells there are 3×10⁴ pg or 30 ng of sharedsequences on the slide. Similarly, there is 10⁴ ×0.5×10⁵ ×6/3×10⁹ pg=1pg of target for the specific sequences. The probe contains 1/2×2 ng or1 ng of shared sequences and 1 ng of specific sequences. Therefore,there is not enough probe to saturate the shared sequences in the targetDNA, but enough to saturate the specific sequences. The signal from theshared sequences is spread at low intensity over the entire genomewhereas the specific signal is concentrated in a compact region. Thus,good contrast can be obtained without adding any blocking genomic DNA atall.

A great deal of genomic DNA can be added to improve the contrast withoutinterfering with the hybridization of the specific sequences, that is, Qremains low even if a great deal of genomic DNA is added.

    Q=10.sup.5 /3×10.sup.9 m.sub.b /m.sub.p =3×10.sup.-5 m.sub.b /m.sub.p.

If a large amount of blocking nucleic acid, for example, 10 ug were used(according to the standard hybridization protocols exemplified inSection VI.B infra wherein the practical limit of total nucleic acid ison the order of 10 ug in a 10 ul hybridization mixture) with the 2 ng ofprobe, then Q=3×10⁻⁵ ×10⁴ ng/2 ng=3/2×10⁻¹ =0.15. Thus, Q is <1, and isso low that the blocking DNA cannot substantially interfere with thedesired signal. Increasing the amount of labeled probe nucleic acid tospeed the hybridization would further decrease Q. In practice, one wouldtypically use 1 ug of blocking DNA for such a hybridization.

Case II. As the size of the target region is increased, the complexityof the probe necessarily is increased, and the amount of DNA in thehybridization mix needs to be increased in order to have a sufficientconcentration of each portion of specific sequence to hybridize. Also,if one desires to decrease the hybridization time of the procedure, theprobe concentration must be increased. In these situations, the increasein probe concentration results in an increase in the amount of sharedsequences in the hybridization mixture, which in turn increases theamount of hybridization that will occur to the shared sequences in thetarget area or areas, thereby reducing the contrast ratio.

With very high complexity probes spanning several entire chromosomes,L/G can approach 1. In order to stain such a portion of the genomewithin a reasonable time, for example, overnight, the concentration oflabeled nucleic acid needs to be increased, for example, 200 ng in 10 ulof hybridization mixture. Up to about 3000 ng of blocking DNA can beused and still keep Q≦5 wherein the calculation is Q=5=0.3 m_(b) /200 ngor m_(b) =1000 ng/0.3=3,333 ng!. In practice, staining 25% and more ofthe human genome (for example, human chromosomes 1, 3 and 4) can beaccomplished with the blocking protocols described below, but thecontrast is less than for that achieved with probes for smaller regions.

III. LabelinQ the Nucleic Acid Fragments of the Heterogeneous Mixture

Several techniques are available for labeling single- anddouble-stranded nucleic acid fragments of the heterogeneous mixture.They include incorporation of radioactive labels, e.g. Harper et al.Chromosoma, Vol 83, pgs. 431-439 (1984); direct attachment offluorochromes or enzymes, e.g. Smith et al., Nucleic Acids Research,Vol. 13, pgs. 2399-2412 (1985), and Connolly et al., Nucleic AcidsResearch, Vol. 13, pgs. 4485-4502 (1985); and various chemicalmodifications of the nucleic acid fragments that render them detectableimmunochemically or by other affinity reactions, e.g. Tchen et al.,"Chemically Modified Nucleic Acids as Immunodetectable Probes inHybridization Experiments," Proc. Natl. Acad. Sci., Vol 81, pgs.3466-3470 (1984); Richardson et al., "Biotin and Fluorescent Labeling ofRNA Using T4 RNA Ligase," Nucleic Acids Research, Vol. 11, pgs.6167-6184 (1983); Langer et al., "Enzymatic Synthesis of Biotin-LabeledPolynucleotides: Novel Nucleic Acid Affinity Probes," Proc. Natl. Acad.Sci., Vol. 78, pgs. 6633-6637 (1981); Brigati et al., "Detection ofViral Genomes in Cultured Cells and Paraffin-Embedded Tissue SectionsUsing Biotin-Labeled Hybridization Probes," Virology, Vol. 126, pgs.32-50 (1983); Broker et al., "Electron Microscopic Visualization of tRNAGenes with Ferritin-Avidin: Biotin Labels," Nucleic Acids Research, Vol.5, pgs. 363-384 (1978); Bayer et al., "The Use of the Avidin BiotinComplex as a Tool in Molecular Biology," Methods of BiochemicalAnalysis, Vol. 26, pgs. 1-45 (1980) Kuhlmann, Immunoenzyme Techniques inCytochemistry (Weinheim, Basel, 1984). Langer-Safer et al., PNAS U.S.A.,79: 4381 (1982): Landegent et al., Exp. Cell Res., 153: 61 (1984); andHopman et al., Exp. Cell Res., 169: 357 (1987).

Exemplary labeling means include those wherein the probe fragments arebiotinylated, modified with N-acetoxy-N-2-acetylaminofluorene, modifiedwith fluorescein isothiocyanate, modified with mercury/TNP ligand,sulfonated, digoxigenenated or contain T-T dimers.

The key feature of "probe labeling" is that the probe bound to thetarget be detectable. In some cases, an intrinsic feature of the probenucleic acid, rather than an added feature, can be exploited for thispurpose. For example, antibodies that specifically recognize RNA/DNAduplexes have been demonstrated to have the ability to recognize probesmade from RNA that are bound to DNA targets Rudkin and Stollar, Nature,265:472-473 (1977)!. The RNA used for such probes is unmodified. Probenucleic acid fragments can be extended by adding "tails" of modifiednucleotides or particular normal nucleotides. When a normal nucleotidetail is used, a second hybridization with nucleic acid complementary tothe tail and containing fluorochromes, enzymes, radioactivity, modifiedbases, among other labeling means, allows detection of the bound probe.Such a system is commerically available from Enzo Biochem (BiobridgeLabeling System; Enzo Biochem Inc., New York, N.Y.).

Another example of a means to visualize the bound probe wherein thenucleic acid sequences in the probe do not directly carry some modifiedconstituent is the use of antibodies to thymidine dimers. Nakane et al.,ACTA HISTOCHEM. CYTOCHEM., 20 (2): 229 (1987), illustrate such a methodwherein thymine-thymine dimerized DNA (T-T DNA) was used as a marker forin situ hybridization. The hybridized T-T DNA was detectedimmunohistochemically using rabbit anti-T-T DNA antibody.

All of the labeling techniques disclosed in the above references may bepreferred under particular circumstances. Accordingly, the above-citedreferences are incorporated by reference. Further, any labelingtechniques known to those in the art would be useful to label thestaining compositions of this invention. Several factors govern thechoice of labeling means, including the effect of the label on the rateof hybridization and binding of the nucleic acid fragments to thechromosomal DNA, the accessibility of the bound probe to labelingmoieties applied after initial hybridization, the mutual compatibilityof the labeling moieties, the nature and intensity of the signalgenerated by the label, the expense and ease in which the label isapplied, and the like.

Several different high complexity probes, each labeled by a differentmethod, can be used simultaneously. The binding of different probes canthereby be distinguished, for example, by different colors.

IV. In Situ Hybridization

Application of the heterogeneous mixture of the invention to chromosomesis accomplished by standard in situ hybridization techniques. Severalexcellent guides to the technique are available, e.g., Gall and Pardue,"Nucleic Acid Hybridization in Cytological Preparations," Methods inEnzymology, Vol. 21, pgs. 470-480 (1981); Henderson, "CytologicalHybridization to Mammalian Chromosomes," International Review ofCytology, Vol. 76, pgs. 1-46 (1982); and Angerer, et al., "In SituHybridization to Cellular RNAs," in Genetic Engineering: Principles andMethods, Setlow and Hollaender, Eds., Vol. 7, pgs. 43-65 (Plenum Press,New York, 1985). Accordingly, these references are incorporated byreferences.

Three factors influence the staining sensitivity of the hybridizationprobes: (1) efficiency of hybridization (fraction of target DNA that canbe hybridized by probe), (2) detection efficiency (i.e., the amount ofvisible signal that can be obtained from a given amount of hybridizationprobe), and (3) level of noise produced by nonspecific binding of probeor components of the detection system.

Generally in situ hybridization comprises the following major steps: (1)fixation of tissue or biological structure to be examined, (2)prehybridization treatment of the biological structure to increaseaccessibility of target DNA, and to reduce nonspecific binding, (3)hybridization of the heterogeneous mixture of probe to the DNA in thebiological structure or tissue; (4) posthybridization washes to removeprobe not bound in specific hybrids, and (5) detection of the hybridizedprobes of the heterogeneous mixture. The reagents used in each of thesesteps and their conditions of use vary depending on the particularsituation.

The following comments are meant to serve as a guide for applying thegeneral steps listed above. Some experimentation may be required toestablish optimal staining conditions for particular applications.

In preparation for the hybridization, the probe, regardless of themethod of its production, may be broken into fragments of the sizeappropriate to obtain the best intensity and specificity ofhybridization. As a general guideline concerning the size of thefragments, one needs to recognize that if the fragments are too longthey are not able to penetrate into the target for binding and insteadform aggregates that contribute background noise to the hybridization;however, if the fragments are too short, the signal intensity isreduced.

Under the conditions of hybridization exemplified in Section VI.Bwherein human genomic DNA is used as an agent to block the hybridizationcapacity of the high copy shared repetitive sequences, the preferredsize range of the probe fragments is from about 200 bases to about 2000bases, more preferably in the vicinity of 1 kb. When the size of theprobe fragments is in about the 800 to about 1000 base range, thepreferred hybridization temperature is about 30° C. to about 45° C.,more preferably about 35° C. to about 40° C., and still more preferablyabout 37° C.; preferred washing temperature range is from about 40° C.to about 50° C., more preferably about 45° C.

The size of the probe fragments is checked before hybridization to thetarget; preferably the size of the fragments is monitored byelectrophoresis, more preferably by denaturing agarose gelelectrophoresis.

Fixatives include acid alcohol solutions, acid acetone solutions,Petrunkewitsch's reagent, and various aldehydes such as formaldehyde,paraformaldehyde, glutaraldehyde, or the like. Preferably,ethanol-acetic acid or methanol-acetic acid solutions in about 3:1proportions are used to fix the chromosomes in metaphase spreads. Forcells or chromosomes in suspension, a fixation procedure disclosed byTrask, et al., in Science, Vol. 230, pgs. 1401-1402 (1985), is useful.Accordingly, Trask et al., is incorporated by reference. Briefly, K₂ CO₃and dimethylsuberimidate (DMS) are added (from a 5× concentrated stocksolution, mixed immediately before use) to a suspension containing about5×10⁶ nuclei/ml. Final K₂ CO₃ and DMS concentrations are 20 mM and 3 mM,respectively. After 15 minutes at 25° C., the pH is adjusted from 10.0to 8.0 by the addition of 50 microliters of 100 mM citric acid permilliliter of suspension. Nuclei are washed once by centrifugation (300g, 10 minutes, 4° C. in 50 mM kCl, 5 mM Hepes buffer, at pH 9.0, and 10mM MgSO₄).

A preferred fixation procedure for cells or nuclei in suspension isdisclosed by Trask et al., Hum. Genet., 78:251-259 (1988), which articleis herein incorporated by reference. Briefly, nuclei are fixed for about10 minutes at room temperature in 1% paraformaldehyde in PBS, 50 mMMgSO₄, pH 7.6 and washed twice. Nuclei are resuspended in isolationbuffer (IB) (50 mM KCl, 5 mM HEPES, 10 mM MgSO₄, 3 mM dithioerythritol,0.15 mg/ml RNase, pH 8.0)/0.05% Triton X-100 at 10⁸ /ml.

Frequently before in situ hybridization chromosomes are treated withagents to remove proteins. Such agents include enzymes or mild acids.Pronase, pepsin or proteinase K are frequently used enzymes. Arepresentative acid treatment is 0.02-0.2 N HCl, followed by hightemperature (e.g., 70° C.) washes. Optimization of deproteinizationrequires a combination of protease concentration and digestion time thatmaximizes hybridization, but does not cause unacceptable loss ofmorphological detail. Optimum conditions vary according to tissue typesand method of fixation. Additional fixation after protease treatment maybe useful. Thus, for particular applications, some experimentation maybe required to optimize protease treatment.

In some cases pretreatment with RNase may be desirable to removeresidual RNA from the target. Such removal can be accomplished byincubation of the fixed chromosomes in 50-100 microgram/milliliter RNasein 2X SSC (where SSC is a solution of 0.15M NaCL and 0.015M sodiumcitrate) for a period of 1-2 hours at room temperature.

The step of hybridizing the probes of the heterogeneous probe mixture tothe chromosomal DNA involves (1) denaturing the target DNA so thatprobes can gain access to complementary single-stranded regions, and (2)applying the heterogeneous mixture under conditions which allow theprobes to anneal to complementary sites in the target. Methods fordenaturation include incubation in the presence of high pH, low pH, hightemperature, or organic solvents such as formamide, tetraalkylammoniumhalides, or the like, at various combinations of concentration andtemperature. Single-stranded DNA in the target can also be produced withenzymes, such as, Exonuclease III van Dekken et al., Chromosoma (Berl)97:1-5 (1988)!. The preferred denaturing procedure is incubation forbetween about 1-10 minutes in formamide at a concentration between about35-95 percent in 2X SSC and at a temperature between about 25°-70° C.Determination of the optimal incubation time, concentration, andtemperature within these ranges depends on several variables, includingthe method of fixation and type of probe nucleic acid (for example, DNAor RNA).

After the chromosomal DNA is denatured, the denaturing agents aretypically removed before application of the heterogeneous probe mixture.Where formamide and heat are the primary denaturing agents, removal isconveniently accomplished by several washes with a solvent, whichsolvent is frequently chilled, such as a 70%, 85%, 100% cold ethanolseries. Alternatively the composition of the denaturant can be adjustedas appropriate for the in situ hybridization by addition of otherconsitutents or washes in appropriate solutions. The probe and targetnucleic acid may be denatured simultaneously by applying thehybridization mixture and then heating to the appropriate temperature.

The ambient physiochemical conditions of the chromosomal DNA and probeduring the time the heterogeneous mixture is applied is referred toherein as the hybridization conditions, or annealing conditions. Optimalhybridization conditions for particular applications can be adjusted bycontrolling several factors, including concentration of theconstituents, incubation time of chromosomes in the heterogeneousmixture, and the concentrations, complexities, and lengths of thenucleic acid fragments making up the heterogeneous mixture. Roughly, thehybridization conditions must be sufficiently close to the meltingtemperature to minimize nonspecific binding. On the other hand, theconditions cannot be so stringent as to reduce correct hybridizations ofcomplementary sequences below detectable levels or to requireexcessively long incubation times.

The concentrations of nucleic acid in the hybridization mixture is animportant variable. The concentrations must be high enough so thatsufficient hybridization of respective chromosomal binding sites occursin a reasonable time (e.g., within hours to several days). Higherconcentrations than that necessary to achieve adequate signals should beavoided so that nonspecific binding is minimized. An important practicalconstraint on the concentration of nucleic acid in the probe in theheterogeneous mixture is solubility. Upper bounds exist with respect tothe fragment concentration, i.e., unit length of nucleic acid per unitvolume, that can be maintained in solution and hybridize effectively.

In the representational examples described in Section VI.B (infra), thetotal DNA concentration in the hybridization mixture had an upper limiton the order of 1 ug/ul. Probe concentrations in the range of 1-20 ng/ulwere used for such whole chromosome staining. The amount of genomicblocking DNA was adjusted such that Q was less than 5. At the low end ofprobe concentration, adequate signals were obtained with a one hourincubation, that is, a time period wherein the probe and blocking DNAare maintained together before application to the targeted material, toblock the high-copy sequences and a 16 hour hybridization. Signals werevisible after two hours of hybridization. The best results (brightsignals with highest contrast) occurred after a 100 hour hybridization,which gave the low-copy target-specific sequences more opportunity tofind binding sites. At the high end of the probe concentration, brightsignals are obtained after hybridizations of 16 hours or less; thecontrast was reduced since more labeled repetitive sequences wereincluded in the probe.

The fixed target object can be treated in several ways either during orafter the hybridization step to reduce nonspecific binding of probe DNA.Such treatments include adding nonprobe, or "carrier", DNA to theheterogeneous mixture, using coating solutions, such as Denhardt'ssolution (Biochem. Biophys. Res. Commun., Vol. 23, pgs. 641-645 (1966),with the heterogeneous mixture, incubating for several minutes, e.g.,5-20, in denaturing solvents at a temperature 5°-10° C. above thehybridization temperature, and in the case of RNA probes, mild treatmentwith single strand RNase (e.g., 5-10 micrograms per millileter RNase) in2X SSC at room temperature for 1 hour).

V. Chromosome-Specific Staining Reagents Comprising Selected Single-CopySequences

V.A. Making and Using a Staining Reagent Specific to Human Chromosome 21

V.A.1. Isolation of Chromosome 21 and Construction of a Chromosome21-Specific Library

DNA fragments from human chromosome-specific libraries are availablefrom the National Laboratory Gene Library Project through the AmericanType Culture Collection (ATCC), Rockville, Md. DNA fragments fromchromosome 21 were generated by the procedure described by Fuscoe etal., in "Construction of Fifteen Human Chromosome-Specific DNA Librariesfrom Flow-Purified Chromosomes," Cytogenet. Cell Genet., Vol. 43, pgs.79-86 (1986), which reference is incorporated by reference. Briefly, ahuman diploid fibroblast culture was established from newborn foreskintissue. Chromosomes of the cells were isolated by the MgSO₄ method ofvan den Engh et al., Cytometery, Vol. 5, pgs. 108-123 (1984), andstained with the fluorescent dyes--Hoechst 33258 and Chromomycin A3.Chromsome 21 was purified on the Lawrence Livermore National Laboratoryhigh speed sorter, described by Peters et al., Cytometry, Vol. 6, pgs.290-301 (1985).

After sorting, chromosome concentrations were approximately 4×10⁵ /ml.Therefore, prior to DNA extraction, the chromosomes (0.2-1.0×10⁶) wereconcentrated by centrifugation at 40,000×g for 30 minutes at 4° C. Thepellet was then resuspended in 100 microliters of DNA isolation buffer(15 mM NaCl, 10 mM EDTA, 10 mM Tris HCl pH 8.0) containing 0.5% SDS and100 micrograms/ml proteinase K. After overnight incubation at 37° C.,the proteins were extracted twice with phenol:chloroform:isoamyl alcohol(25:24:1) and once with chloroform:isoamyl alcohol (24:1). Because ofthe small amounts of DNA, each organic phase was reextracted with asmall amount of 10 mM Tris pH 8.0, 1 mM EDTA (TE). Aqueous layers werecombined and transferred to a Schleicher and Schuell mini-collodionmembrane (#UHO20/25) and dialyzed at room temperature against TE for 6-8hours. The purified DNA solution was then digested with 50 units of HindIII (Bethesda Research Laboratories, Inc.) in 50 mM NaCl, 10 mM Tris HClpH 7.5, 10 mM MgCl₂, 1 mM dithiothreitol. After 4 hours at 37°, thereaction was stopped by extractions with phenol and chloroform asdescribed above. The aqueous phase was dialyzed against water overnightat 40° C. in a mini-collodion bag and then 2 micrograms of Charon 21Aarms cleaved with Hind III and treated with calf alkaline phosphatase(Boehringer Mannheim) were added. This solution was concentrated undervacuum to a volume of 50-100 microliters and transferred to a 0.5 mlmicrofuge tube where the DNA was precipitated with one-tenth volume 3Msodium acetate pH 5.0 and 2 volumes ethanol. The precipitate wascollected by centrifugation, washed with cold 70% ethanol, and dissolvedin 10 microliters of TE.

After allowing several hours for the DNA to dissolve, 1 microliter of10X ligase buffer (0.5M Tris HCl pH 7.4, 0.1M MgCl₂, 0.1Mdithiothreitol, 10 mM ATP, 1 mg/ml bovine serum albumin) and 1 unit ofT4 ligase (Bethesda Research Laboratory, Inc.) were added. The ligationreaction was incubated at 10° C. for 16-20 hours and 3 microliteraliquots were packaged into phage particles using in vitro extractsprepared from E. coli strains BHB 2688 and BHB 2690, described by Hohnin Methods in Enzymology, Vol. 68, pgs. 299-309 (1979) MolecularCloning: A Laboratory Manual, (Cold Spring Harbor Laboratory, New York,1982). Briefly, both extracts were prepared by sonication and combinedat the time of in vivo packaging. These extracts packaged wild-typelambda DNA at an efficiency of 1-5×10⁸ plaque forming units (pfu) permicrogram. The resultant phage were amplified on E. coli LE392 at adensity of approximately 10⁴ pfu/150 mm dish for 8 hours to preventplaques from growing together and to minimize differences in growthrates of different recombinants. The phage were eluted from the agar in10 ml SM buffer (50 mM Tris HCl pH 7.5, 10 mM MgSO₄, 100 mM NaCl, 0.01%gelatin) per plate by gentle shaking at 40° C. for 12 hours. The plateswere then rinsed with an additional 4 ml of SM. After pelleting cellulardebris, the phage suspension was stored over chloroform at 4° C.

V.A.2. Construction and Use of Chromosome 21--Specific Stain forStaining Chromosome 21 of Human Lymphocytes

Clones having unique sequence inserts are isolated by the method ofBenton and Davis, Science, Vol. 196, pgs. 180-182 (1977). Briefly, about1000 recombinant phage are isolated at random from the chromosome21-specific library. These are transferred to nitrocellulose and probedwith nick translated total genomic human DNA.

Of the clones which do not show strong hybridization, approximately 300are picked which contain apparent unique sequence DNA. After theselected clones are amplified, the chromosome 21 insert in each clone is³² P labeled and hybridized to Southern blots of human genomic DNAdigested with the same enzyme used to construct the chromosome 21library, i.e., Hind III. Unique sequence containing clones arerecognized as those that produce a single band during Southern analysis.Roughly, 100 such clones are selected for the heterogeneous mixture. Theunique sequence clones are amplified, the inserts are removed by HindIII digestions, and the inserts are separated from the phage arms by gelelectrophoresis. The probe DNA fragments (i.e., the unique sequenceinserts) are removed from the gel and biotinylated by nick translation(e.g., by a kit available from Bethesda Research Laboratories). LabeledDNA fragments are separated from the nick translation reaction usingsmall spin columns made in 0.5 ml Eppendorph tubes filled with SEPHADEX(trade name for a hydrophilic, insoluble molecular-sieve chromatographicmedium, made by cross-linking dextran) G-50 (medium) swollen in 50 mMTris, 1 mM EDTA, 0.1% SDS, at pH 7.5. Human lymphocyte chromosomes areprepared following Harper et al., Proc. Natl. Acad. Sci., Vol. 78, pgs.4458-4460 (1981). Metaphase and interphase cells were washed 3 times inphosphate buffered saline, fixed in methanol-acetic acid (3:1) anddropped onto cleaned microscope slides. Slides are stored in a nitrogenatmosphere at -20° C.

Slides carrying interphase cells and/or metaphase spreads are removedfrom the nitrogen, heated to 65° C. for 4 hours in air, treated withRNase (100 micrograms/ml for 1 hour at 37° C.), and dehydrated in anethanol series. They are then treated with proteinase K (60 ng/ml at 37°C. for 7.5 minutes) and dehydrated. The proteinase K concentration isadjusted depending on the cell type and enzyme lot so that almost nophase microscopic image of the chromosomes remains on the dry slide. Thehybridization mix consists of (final concentrations) 50 percentformamide, 2X SSC, 10 percent dextran sulfate, 500 micrograms/ml carrierDNA (sonicated herring sperm DNA), and 2.0 microgram/ml biotin-labeledchromsome 21-specific DNA. This mixture is applied to the slides at adensity of 3 microliters/cm² under a glass coverslip and sealed withrubber cement. After overnight incubation at 37° C., the slides arewashed at 45° C. (50% formamide-2XSSC pH 7, 3 times 3 minutes; followedby 2XSSC pH 7, 5 times 2 minutes) and immersed in BN buffer (0.1M Nabicarbonate, 0.05 percent NP-40, pH 8). The slides are never allowed todry after this point.

The slides are removed from the BN buffer and blocked for 5 minutes atroom temperature with BN buffer containing 5% non-fat dry milk(Carnation) and 0.02% Na Azide (5 microliter/cm² under plasticcoverslips). The coverslips are removed, and excess liquid brieflydrained and fluorescein-avidin DCS (3 microgram/ml in BN buffer with 5%milk and 0.02% NaAzide) is applied (5 microliter/cm²). The samecoverslips are replaced and the slides incubated 20 minutes at 37° C.The slides are then washed 3 times for 2 minutes each in BN buffer at45° C. The intensity of biotin-linked fluorescence is amplified byadding a layer of biotinylated goat anti-avidin antibody (5 microgram/mlin BN buffer with 5% goat serum and 0.02% Na Azide), followed, afterwashing as above, by another layer of fluorescein-avidin DCS.Fluorescein-avidin DCS, goat antiavidin and goat serum are all availablecommercially, e.g., Vector Laboratories (Burlingame, Calif.). Afterwashing in BN, a fluorescence antifade solution, p-phenylenediamine (1.5microliter/cm² of coverslip) is added before observation. It isimportant to keep this layer thin for optimum microscopic imaging. Thisantifade significantly reduced fluorescein fading and allows continuousmicroscopic observation for up to 5 minutes. The DNA counterstains (DAPIor propidium iodide) are included in the antifade at 0.25-0.5microgram/ml.

The red-fluorescing DNA-specific dye propidium iodide (PI) is used toallow simultaneous observation of hybridized probe and total DNA. Thefluorescein and PI are excited at 450-490 nm (Zeiss filter combination487709). Increasing the excitation wavelength to 546 nm (Zeiss filtercombination 487715) allows observation of the PI only. DAPI, a bluefluorescent DNA-specific stain excited in the ultraviolet (Zeiss filtercombination 487701), is used as the counterstain when biotin-labeled andtotal DNA are observed separately. Metaphase chromosome 21s are detectedby randomly located spots of yellow distributed over the body of thechromosome.

V.B. Improved Method for Efficiently Selecting Chromosome 21 Single-CopySequences

Fuscoe et al., Genomics, 5:100-109 (1989) provides more efficientprocedures than the method described immediately above (V.A.2) forselecting large numbers of single-copy sequence or very low copy numberrepeat sequence clones from recombinant phage libraries and demonstratestheir use to stain chromosome 21. Said article is hereby incorporated byreference. Briefly, clones were selected from the Charon 21A libraryLL21NS02 (made from DNA from human chromosome 21) using two basicprocedures. In the first, the phage library was screened in two stagesusing methods designed to be more sensitive to the presence ofrepetitive sequences in the clones than the method of Section V.A.2. Theselected clones were then subcloned into plasmids. The 45Q inserts thusselected form the library pBS-U21. The second was in a multistep processin which: 1) Inserts from LL21NS02 were subcloned into Bluescribeplasmids, 2) plasmids were grown at high density in bacterial colonieson nitrocellulose filters and 3) radioactive human genomic DNA washybridized to the plasmid DNA on nitrocellulose filters at lowstringency in two steps and 4) plasmids having inserts that failed tohybridize were selected as potentially carrying single-copy sequences.Fifteen hundred and thirty colonies were picked in this manner to formthe library pBS-U21/1530.

Southern analysis indicated that the second procedure was more effectiveat recognizing repetitive sequence than the first. Fluorescence in situhybridization with DNA from pBS-U21/1530 allowed specific, intensestaining of the number 21 chromosomes in metaphase spreads made fromhuman lymphocytes. Hybridization with pBS-U21 gives less specificstaining of chromosome 21. Details concerning the Fuscoe et al. methodof selecting single-copy sequence or very low repeat sequence probesfrom recombinant libraries follow immediately below.

V.B.1. Phage Screening

Charon 21A phage from the LL21NS02 library were plated onto 30 plates atan average density of 86 pfu per plate. DNA from each plate wastransferred onto HATF085-50 nitrocellulose filters (Benton and Davis,supra) by blotting. Duplicate plaque lifts were prepared from eachplate. One set of filters was hybridized with an oligonucleotide thatspans the cloning site in Charon 21A (Fuscoe, Gene, 52:291, 1987).Hybridization occurs when the Hind III cloning site is not interruptedby an insert. The other set was subjected to the primary phage screenfor human repetitive sequences. Phage that were negative in bothhybridizations (indicating that the phage were recombinant and theamount of repetitive DNA was below the limit of detection) were pickedinto individual wells of microtiter dishes. To assist with theidentification of the appropriate plaques, the agar plates werephotographed onto transparencies at 1:1 magnification. These wereoverlaid on the autoradiographs. The selected phage were then screenedagain using the same procedure.

Primary Phage Screen For Repetitive Sequences: Phage clones containingrepetitive DNA sequences were identified by hybridization with ³²P-labeled human lymphocyte DNA. The DNA was ³² P labeled by nicktranslation Rigby et al., J. Mol. Biol., 113:237 (1977)! to a specificactivity of >10⁸ cpm/ug. Plaque lifts were prepared as above. They wereprehybridized in buffer B (50 ml of 50% formamide, 5x SSC, 5x Denhart'sand 250 ug/ml sonicated, denatured herring sperm DNA) at 42° C. for 3hours and hybridized in 50 ml of buffer B containing 3×10⁶ cpms of probeper ml at 42° C. overnight. The filters were washed once at roomtemperature in 2x SSC, 5x Denhart's, and 0.1% SDS for 10 minutes, twicein the same buffer at 65° C. for 30 minutes each and finally rinsedtwice in 0.1x SSC at room temperature. After air drying, the filterswere exposed to x-ray film (Kodak X-AR5) with an intensifier screen for48 hours at 80° C.

Secondary Phaae Screen for Repetitive Sequences: Recombinant plaquesgiving no hybridization signal with the oligonucleotide or the genomicDNA were picked into individual wells of 96 well microtiter platescontaining 50 μl of SM (100 mM NaCl, 8 mM MgSO₄, 50 mM Tris HCl pH 7.5,0.1% gelatin) and the phage were allowed to diffuse overnight. Fivemicroliters of each phage isolate were transferred to a conical bottommicrotiter tray well and combined with 5 μl of a 1:5 dilution ofstationary phase E. coli LE392, prepared by incubation overnight at 37°C. with good aeration in L-broth containing 0.2% maltose and 10 mMMgSO₄. Infection was performed at 37° C. for 15 minutes with no shaking.Six microliters of each infection were spotted onto a nitrocellulosefilter (Millipore HATF 085 150) that had been soaked in a 1:5 dilutionof an overnight culture of LE392, described above. The filters werehybridized and screened under the conditions of the primary screenfollowing plaque formation at 37° C. Again, plaques showing nohybridization signal were picked for subsequent evaluation.

V.B.2. Subcloning From Charon 21A Phage Into Plasmids

Inserts were subcloned in bulk from Charon 21A into Bluescribe plasmids(Stratagene). Bluescribe is a 2.75 kb, pUC19-derived, high copy numbervector which permits selection on ampicillin and the chromogenicindicator Xgal. Run-off RNA transcripts can be made from T3 and T7promotors that flank the lacZ gene and the cloning site. Charon 21Arecombinant phage and Bluescribe plasmid DNAs were digested tocompletion with Hind III. The Bluescribe DNA was then dephosphorylatedwith calf intenstine alkaline phosphatase (Boehringer Mannheim). Fourhundred ng of Bluescribe vector was ligated to 5 μg of Hind III-cleavedphage DNA with 1 unit ligase Bethesda Research Laboratories (BRL)! in 10μl at 15° C. overnight. E. coli JM83 or DH5alpha (BRL) cells weretransformed with 1 μl of the ligation reaction by the method of Dagertand Ehrlich, Gene, 6:23 (1979). The transformed cells were plated ontoL-plates containing 100 μg/ml ampicillin (L-amp 100) and 40 μg/ml X-gal(BRL) and incubated overnight at 37° C. Recombinants formed paleblue-white colonies and grew larger than the deep blue nonrecombinants.The recombinants were picked into individual wells of 96 well microtiterdishes filled with L-Amp-100. After overnight growth, glycerol was addedto a final concentration of 15%, and the cells were frozen at -80° C.

V.B.3 Plasmid Colony Screening For Repetitive Sequences.

Primary Plasmid Screen For Repetitive Sequences: A microtiter dishinoculating block consisting of 96 machine screws arranged in the 8×12pattern of the wells in a 96 well microtiter dish was used to transferbacteria containing recombinant plasmids to a nitrocellulose filter. Thefilter was overlayed on an L-amp 100 plate and 96 bacterial-plasmidcolonies were grown overnight. The colonies on each filter weresufficiently well separated that the overlap between plasmid DNAs fromadjacent colonies was minimal. The filters were prepared forhybridization as described by Weber et al., Mol. Cell Biol., 8:1137(1987). Briefly the bacteria were lysed with 10% SDS, and the DNA wasdenatured (0.5M NaOH, 1.5M NaCl) and fixed (1M NH₄ OAc, 0.02M NaOH).Bacterial debris was removed by washing in 2×SSC. The filters wereprehybridized and hybridized with ³² P-labeled human genomic DNA at1×10⁶ cpm/ml as in phage screening except that the hybridizationtemperature was 34° C.

Secondary Plasmid Hybridizations Screen For Repetitive Sequences:Colonies negative on the first screen were picked into fresh microtiterdishes as described above. New filters were prepared and wererehybridized as in the primary screen except that the probeconcentration was increased to 3×10⁶ cpm/ml. Colonies negative on thisscreen were picked into new microtiter dishes.

V.B.4 Plasmid Copy Number Quantitation

Twelve clones were picked at random from the pBS-U21 library and used toinoculate a 96-well microtiter dish (100 μl L-broth plus 100 μg/mlampicillin per well). There were 8 identical rows of the 12 individualclones. These were grown overnight at 37° C. One column of 8 cultures,did not grow. All of the 88 colonies that grew were of approximately thesame size. The inoculating block was used to replicate the bacterialarray onto 2 nitrocellulose filters overlaying L-amp 100 plates. Thebacteria on these filters were then incubated for 20 hours, one at 37°C. and one at 32° C. Each filter was then cut in half (40 or 48colonies). One half of each filter was placed into 10 ml L-brothcontaining 100 μg/ml ampicillin. The bacteria were eluted together byvortexing and stored at 4° C. The other half of each filter wastransferred to L-agar plates containing 100 μg/ml chloramphenicol andincubated 24 hours at 37° C. These filters were then placed in 10 mlL-broth containing 100 μg/ml ampicillin, and the bacteria were elutedand stored. The optical density of the eluted bacteria (determined at650 nm) ranged between 3.6 and 5.3. The number of colony forming units(cfu) per filter was determined by plating dilutions onto L-amp 100plates. Finally, plasmid DNA was isolated from 1.5 ml of the elutedbacteria from each filter by the alkaline extraction method Birnboim andDoly, Nucl. Acids Res., 7:1513 (1979)!. The amount of isolated DNA wasmeasured in two ways: 1) Test and known amounts of standard DNA wereseparated using agarose gel electrophoresis. The gels were then stainedwith ethidium bromide and the amount of test DNA was determined bycomparison with the standard. 2) The isolated DNA was stained withHoechst 33258 and the fluorescence intensity (proportional to DNAamount) was measured fluorimetrically.

V.B.5. Southern Hybridization

Southern blot hybridizations were performed using standard proceduresManiatis et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory 1982)!. Briefly, 5 μg or 10 μg of genomic DNA fromcells in a mapping panel was digested to completion with Hind III andsize fractionated on 0.8% agarose minigels. Alternatively, the DNA wasprepared from cells encapsulated in agarose. Approximately 10⁶ cells (-6μg) were mixed with 30 μl of low gelling temperature agarose (SeaPlaque, FMC). The final agarose concentration was 0.5%. After cooling,the plugs were incubated at 50° C. in 0.5M EDTA pH 8.0, 1% sarcosine, 2mg/ml proteinase K for 40 hr. They could then be stored at least 1 yearat 4° C. The plugs were rinsed in 10 volumes 10 mM Tris pH 7.5, 0.1 mMEDTA containing 1 mM phenylmethylsulfonyl fluoride (PMSF) three timesfor 20 min each at room temperature and in this buffer without PMSFtwice for 30 min each. The plugs were transferred to 1× medium saltbutter (Maniatis et al., supra) containing 200 units Hind III and 300μg/ml bovine serum albumin and incubated 3 hr at 37° C. After melting at65° C. for 3 min, the DNA-agarose mixture was placed into wells ofagarose gels for electrophoresis. The size fractionated DNA wastransferred to nitrocellulose and probed with plasmids nick translatedto 10⁸ cpm/ug with ³² P. Hybridization was in 50% formamide, 5×SSC at42° C.

V.B.6. Fluorescence in Situ Hybridization

Biotin labeling of the probe, metaphase spread preparation, andhybridization were as described by Pinkel et al. (I), supra. Briefly DNAsamples from the chromosome 21 probe collections pBS-U21 andpBS-U21/1530 (including plasmid DNA) were biotin labeled by nicktranslation (BRL), and used at a concentration of 4 μg/ml inhybridization mix containing 50% formamide, 2×SSC, 10% dextran sulfate,and 1 mg/ml carrier DNA (herring). The metaphase spreads were treatedwith RNAse (100 μg/ml in 2×SSC) at 37° C. for 1 hour, heated (70° C. in70% formamide, 2×SSC for 2 minutes) to denature the target DNA, anddehydrated in ethanol. The hybridization mix was heated to 70° C. for 5minutes to denature the probe, applied to the slide in a 37° C. glovebox, and sealed under a glass coverslip. The slide was incubated at 37°C. overnight. After incubation, the cover slip was removed and the slidewas washed twice in 50% formamide, 2X SSC at 45° C. for 2 minutes, andonce in 0.1M phosphate buffer (pH 8) containing 0.1% NP40 (PN buffer)for 2 minutes. Fluorescent staining of the hybridized probe wasaccomplished by alternately incubating with fluorescein-avidin DCS andbiotinylated anti-avidin antibody (Vector Laboratories, Burlingame,Calif.), both at 5 μg/ml in PN buffer plus 5% nonfat dry milk, untilthree layers of avidin were applied. These incubations were 20 minutesin duration at room temperature. Slides were washed in PN betweenlayers. The slides were counterstained with propidium iodide (1 μg/ml)in a fluorescence anti-fade solution Johnson and Arovjo, J. Immunol.Methods, 43:349-350, (1981)!.

V.B.7 Library DBS-U21

Phage Screening: Two thousand five hundred phage plaques were screenedtwice to identify recombinant phage carrying unique-sequence inserts. Inthe first screen, 850 out of 2500 plaques were found to benon-recombinant, and 524 recombinants had undetectable levels of humanrepetitive sequences. After the second screen, 450 recombinant phageremained negative. Inserts from these phage were subcloned in bulk intoBluescribe plasmids. Eight hundred and forty recombinant plasmids wereselected to form the library designated pBS-U21.

pBS-U21 Characterization: Plasmid DNA samples from 48 random clones frompBS-U21 were prepared (Birnboin and Doly, supra) and the plasmid insertswere sized on agarose gels. Forty-four clones contained single Hind IIIfragments, three clones contained 2 Hind III fragments and one clonecontained 3 Hind III fragments. The sizes ranged from 4.8 kb to <0.1 kbwith the average being 2.0 kb. Seventy percent of the clones containedinserts of greater than 0.5 kb. Some plasmid clones contained Hind IIIinserts of nearly identical size. To determine if the inserts in theseclones were identical, DNA from each clone was cleaved with the 4-cutterSau3A which should cut the insert into multiple pieces of average sizeabout 250 bases. Agarose gel electrophoresis of these fragmentssuggested that 4 pairs of identical clones were present in the 48tested.

The frequency of unique and repeat-sequences in pBS-U21 was estimated byhybridizing radiolabeled DNA from 18 of the 48 clones against Southernblots of HindIII digested human DNA. Clones producing a single band ofthe predicted size and intensity on an autoradiograph of the blot werescored as carrying a unique-sequence insert. Clones producing multiplebands or smears were scored as carrying repetitive inserts.

The average size of the 18 inserts was 2.9 kb.

Eleven clones hybridized to unique single Hind III fragments in thehuman DNA of the same size as the cloned insert. The remaining 7 showedthe presence of repetitive sequences. Thus about 60% (11/18) clones inpBS-U21 proved to carry unique sequence inserts.

Detection of Residual Repetitive Sequences in pBS-U21 by Plasmid ColonyScreening: The 48 plasmid clones from pBS-U21 described above wererescreened by plasmid colony screening. Sixteen were found to berepetitive. Eighteen of the 48 clones were those characterized bySouthern analysis as described above. One third of these (7/18) wereshown to contain repetitive sequences by Southern analysis. Plasmidcolony screening detected 5 of the 7 repeats. Thus only about 10% (2/18)of the clones that passed the plasmid colony screen contained repetitivesequences.

Sensitivity of Plasmid Colony Screening: The sensitivity of the plasmidcolony screen is due, at least in part, to the large amount and highdensity of human DNA in the plasmid colonies. Colonies grown overnightat 37° C. (the condition used for colony production in the colonyscreening assay) contained about 6×10⁸ bacteria/colony and about 2.6 ugplasmid DNA per colony. This would suggest that each bacteria containedabout 1000 plasmids if each plasmid was about 5 kb in size. This is inthe range expected for the pUC series of plasmids. The bacterial growthconditions can affect the plasmid density significantly. For example,bacteria grown at 32° C. contain about 5 times fewer plasmids. Theplasmid DNA density can be increased by treatment with chloramphenicolalthough this was not used for colony production in this example. Themost plasmid DNA per colony was obtained by 20 hr growth at 37° C.followed by 24 hr growth on chloramphenicol at 37° C.

V.B.8. Library pBS-U21/1530:

Plasmid Selection: Inserts from the entire LL21NS02 library weresubcloned into Bluescribe. Ten thousand seven hundred and fifty tworecombinants were individually picked into the wells of microtiterplates. Half of these were subjected to the primary plasmid colonyscreen. The negative clones (2450) were transferred to a new set ofplates and subjected to the secondary plasmid colony screen as describedabove. The negative clones (1530) were transferred to a new set ofplates and are being maintained individually. The collection of suchclones form the library pBS-U21/1530.

pBS-U21/1530 Characterization: Plasmid DNA samples from 46 random clonesfrom pBS-U21/1530 were prepared (Birnboim and Doly, supra), and theplasmid inserts were sized on agarose gels. Forty-four of these clonescontained a single insert while two contained 2 inserts. The insertsranged from 4 kb to <0.25 kb. The average insert size was 0.9 kb. Halfof the inserts were larger than 0.25 kb.

DNA samples from 13 of the 46 random clones were characterized bySouthern analysis using Hind III digested DNA from human cells asdescribed above. These 13 clones carried 14 inserts. About 80% of theclones (10/13) proved to be unique sequence.

V.B.9 In Situ Hybridization with pBS-U21 and pBS U21/1530

Both pBS-U21 and pBS-U21/1530 preferentially and distinctly stained thenumber 21 chromosome. The pBS-U21 library showed preferentialhybridization to both copies of chromosome 21, but there was substantialhybridization to other chromosomes. The hybridization of pBS-U21/1530was more specific. The hybridization was weak on the short arm of thechromosome where the repetitive ribosomal sequences reside; theintensity of the signal was concentrated in the middle of the long armof chromosome 21. Many interphase nuclei showed distinct labeling of twoapparent chromosome 21 domains. The fact that the pBS-U21 librarycontained more repeat-sequence inserts that might hybridize nonspecifically than did the pBS-U21/1530 library may account for thehigher specificity of the pBS-21/1530 hybridization.

V.B.10 Conclusion

Demonstrated in this subsection is that bulk subcloning of the humanchromosome 21 library LL21NS02 into plasmids, followed by colonyhybridization with radiolabeled human genomic DNA, allows efficientselection of a library in which 80 to 90% of the clones contain onlyunique sequences. Also shown is that DNA from clones selected by thisprocedure can be used in fluorescence in situ hybridization tospecifically stain the number 21 chromosomes in human metaphase spreadsand interphase nuclei. The intensity of staining of the unique sequenceprobes is not as high as that obtained using highly repeated DNAsequences as probes Pinkel et al. (I), supra! because the amount oftarget sequence for the unique sequence probes is relatively low and isspread out over an extended region. The amount of target sequence for ahighly repeated probe may be around 5 Mb; whereas the amount of targetsequence for the DNA probes isolated from the pBS-U21 and pBS-U21/1530libraries are only about 0.9 Mb and 1.3 Mb, respectively. In addition,the high complexity of the probes means that each element is present atlow concentration during the hybridization. The nonspecifichybridization with DNA from pBS-U21/1530 may be due to the remaining lowlevel repeats or to unique sequences that are not from chromosome 21.Some non-specific hybridization may also result from the binding oflabeled plasmid sequences.

The LL21NS02 library appears to be highly chromosome-specific in thatabout 80% of the clones from pBS-U21 and 70% of the clones frompBS-U21/1530 map to chromosome 21.

Further concluded herein was that in a comparison of the ability ofphage plaque screening and plasmid colony screening to remove clonescontaining repetitive DNA, the plasmid colony screen was significantlymore sensitive than the phage plaque screen. The higher sensitivity ofthe plasmid colony screen resulted from the high density of human DNA inthe plasmids of a colony. The human DNA content of the plasmid colonywas calculated to be about 5×10⁴ times that of a plaque, whereas thehuman DNA density of a plasmid colony was about 4×10³ times that of aplaque.

These experiments indicated that bulk subcloning of lambda phagelibraries into plasmids followed by plasmid colony screening asdescribed above should result in selection of a plasmid library in whichapproximately 90% of the plasmids carry unique-sequence inserts. Sometechnical improvement in the colony screening procedure, such as the useof chloramphenicol to increase the plasmid content of the bacteria, maymake it possible to obtain a positive hybridization signal fromessentially every colony, thus improving sensitivity. Using controlcolonies with well characterized inserts, it should be possible tochoose upper and lower intensity thresholds that would optimizeselection of unknown clones containing inserts with the desiredcharacteristics.

V.C. Hybridization with a Collection of Chromosome 4 Single-CopySequences

Chromosome 4 Single-Copy Sequences. One hundred and twenty clonescarrying chromosome 4-specific single-copy sequence inserts selectedfrom the Charon 21A library LL04NS01 (ATCC accession number 57700; VanDilla et al., supra; see Table 1) were supplied by C. Gilliam (HarvardUniversity) Gilliam et al., Nucleic Acids Res., 15:1445 (1987)!. Thehuman inserts were all about 3 kilobases (kb) in length, so the ratio ofinsert to vector DNA was <0.1. Total phage DNA was produced from eachclone individually using DEAE-cellulose columns (Whatman DE-52) Helms etal., DNA, 4:39 (1985)!. DNA pooled from the 120 clones was biotinylatedby nick-translation with biotin-11-dUTP (Bethesda Research Laboratories)and recovered at a concentration of about 20 nanograms per microliter(ng/μl) using Sephadex G-50 spin columns.

Cells. Metaphase spreads from human lymphocytes were prepared frommethotrexate-synchronized cultures by using the procedure of Harper etal., supra. The cells were fixed in methanol/acetic acid, 3:1. Slideswere stored at -20° C. in plastic bags filled with nitrogen gas.

In Situ Hybridization: Single-Copy Hybridization. Hybridization wasaccomplished by using a modification of the procedure described byPinkel et al., PNAS USA. 83: 2934 (1986). The slide mounted cells weretreated with RNase 100 micrograms per milliliter (μg/ml) in 0.3 molar(M) sodium chloride (NaCl)/30 millimolar (mM) sodium citrate at 37° C.for 1 hr), dehydrated in a 70%/85%/100% ethanol series, treated withproteinase K (0.3-0.6 μg/ml in 20 mM Tris/2 mM CaCl₂, pH 7.5, for 7.5min at 37° C.), and fixed 4% paraformaldehyde in phosphate-bufferedsaline (PBS; in g/liter, KCl, 0.2; KH₂ PO₄, 0.2; NaCl, 8; Na₂ HPO₄ ·7H₂O, 2.16) plus 50 mM MgCl₂ for 10 min at room temperature!. The DNA inthe target cells was denatured by immersion in 70% formamide/2XSSC (0.3MNaCl/30 mM sodium citrate) at pH 7, for 2 min at 70° C. Thehybridization mixture 10 μl total volume consisting of 50% formamide,0.3M NaCl/30 mM sodium citrate (final concentration), 10% dextransulfate, 50 μg of sonicated herring DNA per ml, and 3-6 ng ofbiotinylated chromosome 4 unique sequences (40-80 ng of total phageDNA)! was then denatured (70° C. for 5 min) and applied. Hybridizationwas at 37° C. overnight (16 hr). Slides were washed in three changes of50% formamide/0.3M NaCl/30 mM sodium citrate (final concentration), pH7, at 45° C. for 5 min each and once in PN buffer (a mixture of 0.1MNaH₂ PO₄ and 0.1M Na₂ HPO₄ to give pH 8/0.1% Nonidet P-40). The slideswere then treated with alternating layers of fluoresceinated avidin andbiotinylated goat antiavidin, both at 5 μg/ml in PNM buffer (PNbuffer/5% non-fat dry milk/0.02% sodium azide, centrifuged to removesolids), for 20 min each at room temperature until three layers ofavidin were applied. The avidin and goat anti-avidin treatments wereseparated by three washes of 3 min each in PN buffer avidin (DCS grade)and anti-avidin from Vector Laboratories (Burlingame, Calif.)!. Afterthe final avidin treatment, a fluorescence antifade solution Johnson andNoqueria, J. Immunol. Methods. 43: 349 (1981)! containing 1 μg of4',6-amidino-2-phenylindole or propidium iodide per ml was applied as acounterstain (1.5 μl/cm² under a no. 1 coverslip).

Results. As shown in FIG. 4H, individual hybridization sites could belocated to within a fraction of the width of a chromatid after overnighthybridization (16 hr) and application of three layers of avidin.Analysis of three spreads from the hybridization with the 120 uniquesequence probes at a total probe concentration of 1.5 pg/ul per kilobaseof human insert, showed 222 fluorescent spots out of the 1440 possibleon the number 4 chromosomes (120 target sites per chromatid×4 chromatidsper metaphase×3 metaphases). Thus, the hybridization efficiency was 15%.There were 814 total spots on all of the chromosomes giving ahybridization specificity of 27%. The experiment demonstrates thatsubstantial hybridization can occur with single copy probes at low probeconcentrations in overnight hybridizations. The contrast ratio ofchromosome 4 relative to the rest of the ##EQU1## (Chromosome 4comprises about 6% of the genome.)

VI. Incapacitating Shared Repetitive Sequences

VI.A. Chromosome 21-Specific Staining Using Blocking DNA

High concentrations of unlabeled human genomic DNA and lambda phage DNAwere used to inhibit the binding of repetitive and vector DNA sequencesto the target chromosomes. Heavy proteinase digestion and subsequentfixation of the target improved access of probes to target DNA.

Human metaphase spreads were prepared on microscope slides with standardtechniques and stored immediately in a nitrogen atmosphere at -20° C.

Slides were removed from the freezer and allowed to warm to roomtemperature in a nitrogen atmosphere before beginning the stainingprocedure. The warmed slides were first treated with 0.6 microgram/mlproteinase K in P buffer (20 mM Tris, 2 mM CaCl₂ at pH 7.5) for 7.5minutes, and washed once in P buffer. The amount of proteinase K usedneeds to be adjusted for different batches of slides. After denaturingthe slides were stored in 2XSSC. A hybridization mix was prepared whichconsisted of 50% formamide, 10% dextran sulfate, 1% Tween 20, 2XSSC, 0.5mg/ml human genomic DNA, 0.03 mg/ml lambda DNA, and 3 microgram/mlbiotin labeled probe DNA. The probe DNA consisted of the highest densityfraction of phage from the chromosome 21 Hind III fragment library (ATCCaccession number 57713), as determined by a cesium chloride gradient.(Both insert and phage DNA of the probe were labeled by nicktranslation.) The average insert size (amount of chromosome 21 DNA), asdetermined by gel electrophoresis was about 5 kilobases. No attempt wasmade to remove repetitive sequences from the inserts or to isolate theinserts from the lambda phage vector.

The hybridization mix was denatured by heating to 70° C. for 5 minutesfollowed by incubation at 37° C. for 1 hour. The incubation allows thehuman genomic DNA and unlabeled lambda DNA in the hybridization mix toblock the human repetitive sequences and vector sequences in the probe.

The slide containing the human metaphase spread was removed from the2XSSC and blotted dry with lens paper. The hybridization mix wasimmediately applied to the slide, a glass cover slip was placed on theslide with rubber cement, and the slide was incubated overnight at 37°C. Afterwards preparation of the slides proceeded as described inSection V.B. (wherein chromosome 21 DNA was stained with fluorescein andtotal chromosomal DNA counterstained with DAPI). FIGS. 1A-C illustratethe results. FIG. 1A is a DAPI image of the human metaphase spreadobtained with a computerized image analysis system. It is a binary imageshowing everything above threshold as white, and the rest as black. Theprimary data was recorded as a gray level image with 256 intensitylevels. (Small arrows indicate the locations of the chromosome 21s.)FIG. 1B is a fluorescein image of the same spread as in FIG. 1A, againin binary form. (Again, small arrows indicate the locations of thechromosome 21s.) FIG. 1C illustrates the positions of the chromosome 21safter other less densely stained objects were removed by standard imageprocessing techniques.

VI.B. Detection of Trisomy 21 and Translocations of Chromosome 4 UsingBluescribe Plasmid Libraries

As illustrated in Section VI.A., a human chromosome-specific library,including its shared repetitive sequences, can be used to stain thatchromosome if the hybridization capacity of the shared repetitivesequences is reduced by incubation with unlabeled human genomic DNA. InSection VI.A., the nucleic acid sequences of the heterogeneous mixturewere cloned in the phage vector Charon 21A, in which the ratio of insertof vector DNA is about 0.1 (4 kb average insert to 40 kb of vector). Inthis section, we demonstrate that transferring the same inserts to asmaller cloning vector, the about 3 kb Bluescribe plasmid, whichincreases the ratio of insert to vector DNA to 0.5, improved thespecificity and intensity of the staining.

As previously discussed, incubation of the probe can be carried out withthe probe alone, with the probe mixed with unlabeled genomic DNA, andwith the probe mixed with unlabeled DNA enriched in all or some sharedrepetitive sequences. If unlabeled genomic DNA is added, then it isimportant to add enough to incapacitate sufficiently the sharedrepetitive sequences in the probe. However, the genomic DNA alsocontains unlabeled copies of the sequences, the hybridization of whichis desired. As explained above, Q is herein defined as the ratio ofunlabeled to labeled copies of the chromosome-specific sequences in thehybridization mixture.

Cells. Metaphase spreads from human lymphocytes were prepared frommethotrexate-synchronized cultures by using the procedure of Harper etal. supra. These and all other cells used in this example were fixed inmethanol/acetic acid, 3:1. Other human lymphocyte cultures wereirradiated with ⁶ Co gamma rays and stimulated with phytohemagglutnin.Colcemid was added 48 hr after stimulation and metaphase spreads wereprepared 4 hr later. Metaphase spreads and interphase cells fromlymphoblastoid cells (GM03716A; Human Mutant Cell Repository, Camden,N.J.) carrying trisomy 21 were prepared after a 4-hr colcemid block.Interphase cells from the cell line RS4;11 carrying t(4;11) andisochromosome 7q were harvested, fixed in methanol/acetic acid, anddropped onto slides Strong et al., Blood, 65:21 (1985)!. Slides werestored at -20° C. in plastic bags filled with nitrogen gas.

pBS-4. The entire chromosome 4 library LL04NS02 (ATCC accession number57745; Van Dilla et al., supra) was subcloned into Bluescribe plasmids(Stratagene La Jolla, Calif.) to form the library pBS-4. The averageinsert to vector DNA ratio in pBS-4 is about 1. The plasmid library wasamplified in bulk and the DNA was extracted using DEAE-cellulose columns(Whatman DE-52) Helms et al., DNA, 4:39 (1985)!. The DNA was thenbiotinylated by nick translation with biotin-11-dUTP (Bethesda ResearchLaboratories) and recovered at a concentration of about 20 ng/ul usingSephadex G-50 spin columns. In some experiments, the biotinylated DNAwas concentrated by ethanol precipitation to achieve higher probeconcentrations.

pBS-21. The entire chromosome 21 library LL21NS02 (ATCC accession number57713; Van Dilla et al., supra) was subcloned into Bluescribe plasmidsto form the library pBS-21. This library was amplified and biotinylatedas described above for pBS-4.

Human genomic DNA. Placental DNA (Sigma) was treated with proteinase K,extracted with phenol, and sonicated to a size range of 200-600 basepairs (bp).

Whole Library Hybridization. Hybridization was as above in section V.Cexcept that RNase, proteinase K, and paraformaldehyde were not used. Theamount of probe and genomic DNA in the hybridization mixture and thelength of the hybridization varied as described in Results. All probeconcentrations refer to the human insert DNA unless otherwise noted. DNAconcentrations were determined by fluorometric analysis (HoefferScientific Instruments, San Francisco). Incubation of the hybridizationmixture prior to hybridization followed two different protocols asindicated immediately below.

Protocol I. The hybridization mixture (10 μl) contained 10-150 ng ofbiotinylated human DNA (20-300 ng of total plasmid DNA) and 0-10 ug ofunlabeled genomic DNA. The mixture was heated to denature the DNA andincubated at 37° C. for a time t before it was added to the slide.Hybridization times ranged from 2 to 110 hr.

Protocol II. Protocol II was identical to Protocol I except that anadditional aliquot of freshly denatured genomic DNA was added to thehybridization mixture after an incubation time t. The mixture was thenincubated an additional time t prior to starting the hybridization. Thevolume of the hybridization mixture was increased <20% by the additionalgenomic DNA.

Microscopy. Quantitative fluorescence measurements were performed usinga video camera on the microscope and a digital image processing system,Trask et al., Human Genet., 78:251 (1988)!

Results. FIG. 4A shows hybridization of pBS-4 to a human metaphasespread with a probe concentration of 1 ng/μl. No genomic DNA was usedand the hybridization mixture was applied immediately afterdenaturation. All of the chromosomes are stained, except near manycentromeres, with two copies of chromosome 4 being stained most heavily.All the chromosomes are stained along most of their lengths due tosequences in the probe which are shared with other chromosomes.Unstained regions, noted by arrows, show locations for which homologoussequences are not present in pBS-4. The unstained regions are mostlycentromeric and along the long arm of the Y chromosome. Blocks ofrepetitive DNA specific to those sites are known to exist.

The visible contrast on chromosome 4 is the result of the interaction ofseveral factors. (i) All of the DNA in chromosome 4 is potential targetfor sequences in the probe, whereas only those sequences on the otherchromosomes that are shared with chromosome 4 can bind probe. (ii) Thehybridization time and probe concentration were high enough to allowsignificant binding of the specific sequences in the probe. (iii) Theratio of probe to target sequences is higher for the specific sequencesthan for the shared sequences Ten nanograms of chromosome 4 DNA washybridized to about 200 ng of human DNA target (4×10⁴ cells), 13 ng ofwhich is chromosome 4. Thus, the ratio of probe to target for thespecific sequences was about 1, whereas for the shared sequences it wasabout 0.05.!

The contrast can be increased by allowing the denatured probe DNA topartially reassociate prior to adding it to the slide, preferentiallydepleting the single-stranded high-copy (predominantly the shared)sequences in the probe Cantor & Schimmel!, Biophysical Chemistry: TheBehavior of Biological Macromolecules, (part III, p. 1228) (Freeman1980)!. A significant increase in staining specificity resulting fromprobe reassociation was observed experimentally for chromosome 4 using ahybridization mixture with 1 ng of probe per microliter (μl) and a 24-hrincubation at 37° C. prior to in situ hybridization (not shown).Likewise, hybridization after a 24 hr incubation of 4 ng of chromosome21 probe per μl resulted in a substantial contrast ratio. That resultindicates that at such concentrations the chromosome-specific sequencesremain substantially single stranded for times on the order of days inthe hybridization mixture. It also demonstrates that other mechanismsthat might inactivate the probe are not significant during theincubation.

FIGS. 4B and 4C show the result of a protocol I hybridization 0.8 ng ofprobe per μl and 24 ng of genomic DNA per μl (Q=2); 1-hr probeincubation and 110-hr hybridization!. Quantitative image analysis showsthat the intensity per unit length of the FITC fluorescein on chromosome4 is approximately 20 times that of the other chromosomes, that is thecontrast ratio is 20:1. Two layers of avidin-fluorescein isothiocyanatehave been used here to make the non-target chromosomes sufficientlybright to be measured accurately. However, the number 4 chromosomes canbe recognized easily after a single layer.

FIG. 4D demonstrates detection of a radiation-induced translocationinvolving chromosome 4 in human lymphocytes protocol I, 1 ng of probeper μl and 76 ng of genomic DNA per μl (Q=5); 1-hr probe incubation and16-hr hybridization!. The contrast ratio was about 5. The hybridizationintensity and specificity shown in FIG. 4D are such that even smallportions of the involved chromosome can be detected.

The ease with which translocations can be recognized offers theopportunity for translocation detection by automated means, such as,computerized microscopy or flow cytometry. See Section VIII infra forelaboration concerning automated detection means.!

FIG. 4E shows that the normal and two derivative chromosomes resultingfrom the translocation between chromosomes 4 and 11 t(4;11)! in cellline RS4;11 can be detected in interphase nuclei as three distinctdomains protocol I, 13.5 ng of probe per μl and 800 ng of genomic DNAper μl (Q=5); 1-hr probe incubation and 16-hr hybridization!. Theincreased probe concentration resulted in brighter signals relative toFIG. 4D. Approximately half of the cells clearly show the presence ofthree nuclear domains, presumably produced by the two portions of theinvolved chromosome 4 and the intact normal chromosome. The domains inthe other nuclei may have been obscured by the nuclear orientation inthese two-dimensional views, by nuclear distortion that occurred duringslide preparation, or because the domains were too close to each otherto be distinguished. Hybridization using procedures that preservethree-dimensional morphology may resolve these issues and also permitgeneral studies of chromosomal domains in interphase nuclei Trask etal., Hum. Genet., 78: 251 (1988)!.

Hybridization of pBS-21 to a metaphase spread from a cell line withtrisomy 21 is shown in FIG. 4F protocol II, 4 ng of probe per μl and 250ng of genomic DNA per μl; 3-hr incubation, additional 250 ng of genomicDNA per μl (Q=1+1); 3-hr probe incubation and 16-hr hybridization!. Asmall amount of hybridization is visible near the centromeres of theother acrocentric chromosomes.

FIG. 4G shows two interphase nuclei from the same hybridization whichclearly show the three chromosome 21 domains. Hybridization with probeprepared according to protocol I resulted in higher relative intensityof the shared signals on the D- and G-group chromosomes, andconsequently it was more difficult to determine the number of number 21chromosomes in interphase (not shown). Increasing stringency by using ahybridization mixture with 55% formamide and 0.15M NaCl/15 mM sodiumcitrate, which lowers the melting temperature about 8° C., did notreduce the unwanted hybridization. Addition of unlabeled pA ribosomalDNA Erikson et al., Gene, 16:1 (1981)! also was ineffective atincreasing specificity.

The centromeric region of the D- and G-group chromosomes containribosomal Erikson et al., id! and alpha satellite sequences and perhapsothers Choo et al., Nucleic Acids Res., 16: 1273 (1988)!. These arerelatively low copy sequences shared with only a few chromosomes, soProtocol I is not very effective at suppressing them relative to thechromosome 21-specific sequences. In addition, these sequences areclustered on the chromosomes, so that even much reduced hybridization isclearly visible. This is especially distracting in analysis ofinterphase nuclei. Calculations indicate that addition of severalaliquots of freshly denatured genomic DNA periodically during theincubation (protocol II) should increase the staining specificity. FIG.4F shows a protocol II hybridization, using two aliquots of genomic DNA,to a metaphase spread from a trisomy 21 cell line. Intense hybridizationto the three number 21 chromosomes is clearly visible and hybridizationto the other D- and G-group chromosomes has been reduced to anacceptable level. FIG. 4G shows that hybridization to chromosomes otherthan chromosome 21 is sufficiently low that the three chromosome 21domains are clearly visible in interphase nuclei. In practice, the mostconvenient procedure for suppressing the shared acrocentrichybridization might be inclusion of unlabeled DNA from one of the otherD- or G-group chromosome libraries (or unlabeled cloned DNA from justthese sequences, if available) as additional competitor. The use oflibraries from non-target chromosomes as blocker for a probe may ingeneral improve contrast. The specific sequences in the probe will notbe blocked (Q=0) no matter how much competitor for the shared sequencesis added.

VI.C. Hybridization of Yeast Artificial Chromosomes (YACS) to HumanMetaDhase Spread

YACS. Seven yeast clones HY1, HY19, HY29, HYAI1.A2, HYA3.A2, HYA3.A9,and HYA9.E6 were obtained from D. Burke (Washington University, St.Louis, Mo.). The lengths of the human DNA in the clones ranged fromabout 100 kb to about 600 kb. Gel electrophoresis was performed toverify the size of these inserts. Each of these clones was grown up andtotal DNA was isolated. The isolated DNA was biotinylated by nicktranslation so that 10-30% of the thymidine was replaced bybiotin-11-dUTP. The concentration of the total labeled DNA after nicktranslations is in the range of 10-20 ng/μl.

Blocking DNA. Human placental DNA (Sigma) was treated with proteinase Kand extracted with phenol and sonicated to a size range of 200-600 bp.Total DNA isolated from yeast not containing an artificial chromosomewas sonicated to a similar size range. Both of these DNA's weremaintained at a concentration of 1-10 μg/μl.

Fluorescence in situ hybridization (FISH). Hybridization followed theprocedures of Pinkel et al. (1988), supra (as exemplified in Sections Vand VI, supra) with slight modifications. Metaphase spreads wereprepared from methotrexate synchronized cultures according to theprocedures of Harper et al. PNAS (USA) 78: 4458-4460, (1981). Cells werefixed in methanol/acetic acid, fixed (3:1), dropped onto slides, airdried, and stored at -20° C. under nitrogen gas until used. The slideswere then immersed two minutes in 70% formamide/2×SSC to denature thetarget DNA sequences, dehydrated in a 70-85-100% ethanol series, and airdried. (SSC is 0.15M NaCl/0.015M Na Citrate, pH 7). Ten - 100 ng ofbiotinylated yeast DNA, and approximately 1 μg each of unlabeled yeastand human genomic DNA were then added to the hybridization mix (finalvolume 10 μl, final composition 50% formamide/2×SSC/10% dextransulfate), heated to 70° C. for 5 min., and then incubated at 37° C. for1 hr to allow the complementary strands of the more highly repeatedsequences to reassociate.

The hybridization mixture was then applied to the slide (approximately 4cm² area) and sealed with rubber cement under a glass cover slip. Afterovernight incubation at 37 ° C. the coverslip was removed and the slidewashed 3 times 3 min each in 50% formamide/2×SSC at 42°-45° C., and oncein PN buffer mixture of 0.1M NaH₂ PO₄ and 0.1M Na₂ HPO₄ to give pH 8;0.1% Nonidet P-40 (Sigma)!. The bound probe was then detected withalternating 20 min incubations (room temperature in avidin-FITC andgoat-anti-avidin antibody, both at 5 μg/ml in PNM buffer (PN buffer plus5% nonfat dry milk, centrifuged to remove solids; 0.02% Na azide).Avidin and anti-avidin incubation were separated by 3 washes of 3 mineach in PN buffer. Two or three layers of avidin were applied (Avidin,DCS grade, and biotinylated goat-anti-avidin are obtained from VectorLaboratories Inc., Burlingame, Calif.).

FIG. 5 shows the hybridization of HYA3.A2 (580 kb of human DNA) to12q21.1. The location of the hybridization was estabilished by using aconventional fluorescent banding technique employing theDAPI/actinomycin D procedure: Schweizer, "Reverse fluorescent chromosomebanding with chromomycin and DAPI," Chromosoma, 58: 307-324 (1976). Thehybridization signal forms a band across the width of each of thechromosome 12s, indicating the morphology of the packing of DNA in thatregion of the chromosome. The YAC clone positions are attributed asshown in Table 2 below.

                  TABLE 2                                                         ______________________________________                                        YAC Competition Hybridization                                                 YAC Clone   Insert Size     Localization                                      ______________________________________                                        HY1         120             Xq23                                              HY19        450             8q23.3                                                                        21q21.1                                           HY29        500             14q12                                             HYA1.A2     250             6q16                                              HYA3.A2     580             12q21.1                                           HYA3.A9     600             14q21                                             HYA9.E6     280             1p36.2                                                                        3q22                                              ______________________________________                                    

VI.D. Hybridization With Human/Hamster Hybrid Cell

Essentially the same hybridization and staining conditions were used inthis example as for those detailed in the procedure of Pinkel et al.(1988), supra and exemplified in Sections V.C. and VI.B., supra. In thisexample, 400 ng of biotin labeled DNA from a hamster-human hybrid cellthat contains one copy of human chromosome 19 was mixed with 1.9 μg ofunlabeled human genomic DNA in 10 μl of hybridization mix. Hybridizationwas for approximately 60 hours at 37° C. Fluorescent staining of thebound probe and counterstaining of the chromosomes was as in the otherexamples above. FIG. 6 shows the results of the hybridization.

VII. Specific Applications.

The present invention allows microscopic and in some cases flowcytometric detection of genetic abnormalities on a cell by cell basis.The microscopy can be performed entirely by human observers, or includevarious degrees of addititional instrumentation and computationalassistance, up to full automation. The use of instrumentation andautomation for such analyses offers many advantages. Among them are theuse of fluorescent dyes that are invisible to human observers (forexample, infared dyes), and the opportunity to interpret resultsobtained with multiple labeling methods which might not besimultaneously visible (for example, combinations of fluorescent andabsorbing stains, autoradiography, etc.) Quantitative measurements canbe used to detect differences in staining that are not detectable byhuman observers. As is described below, automated analysis can alsoincrease the speed with which cells and chromosomes can be analysed.

The types of cytogenetic abnormalities that can be detected with theprobes of this invention include: Duplication of all or part of achromosome type can be detected as an increase in the number or size ofdistinct hybridization domains in metaphase spreads or interphase nucleifollowing hybridization with a probe for that chromosome type or region,or by an increase in the amount of bound probe. If the probe is detectedby fluorescence, the amount of bound probe can be determined either flowcytometrically or by quantitative fluorescence microscopy. Deletion of awhole chromosome or chromosome region can be detected as a decrease inthe number or size of distinct hybridization domains in metaphasespreads or interphase nuclei following hybridization with a probe forthat chromosome type or region, or by a decrease in the amount of boundprobe. If the probe is detected by fluorescence, the amount bound can bedetermined either flow cytometrically or by quantitative fluorescencemicroscopy. Translocations, dicentrics and inversions can be detected inmetaphase spreads and interphase nuclei by the abnormal juxtaposition ofhybridization domains that are normally separate following hybridizationwith probes that flank or span the region(s) of the chromosome(s) thatare at the point(s) of rearrangement. Translocations involve at leasttwo different chromosome types and result in derivative chromosomespossessing only one centromere each. Dicentrics involve at least twodifferent chromosome types and result in at least one chromosomefragment lacking a centromere and one having two centromeres. Inversionsinvolve a reversal of polarity of a portion of a chromosome.

VII.A Banding Analysis

Substantial effort has been devoted during the past thirty years todevelopment of automated systems (especially computer controlledmicroscopes) for automatic chromosome classification and aberrationdetection by analysis of metaphase spreads. In recent years, effort hasbeen directed at automatic classification of chromosomes which have beenchemically stained to produce distinct banding patterns on the variouschromosome types. These efforts have only partly succeeded because ofthe subtle differences in banding pattern between chromosome types ofapproximately the same size, and because differential contraction ofchromosomes in different metaphase spreads causes a change in the numberand width of the bands visible on chromosomes of each type. The presentinvention overcomes these problems by allowing construction of reagentswhich produce a staining pattern whose spacing, widths and labelingdifferences (for example different colors) are optimized to facilitateautomated chromosome classification and aberration detection. This ispossible because hybridization probes can be selected as desired alongthe lengths of the chromosomes. The size of a band produced by such areagent may range from a single small dot to a substantially uniformcoverage of one or more whole chromosomes. Thus the present inventionallows construction of a hybridization probe and use of labeling means,preferably fluorescence, such that adjacent hybridization domains can bedistinguished, for example by color, so that bands too closely spaced tobe resolved spatially can be detected spectrally (i.e. if red and greenfluorescing bands coalesce, the presence of the two bands can bedetected by the resulting yellow fluorescence).

The present invention also allows construction of banding patternstailored to particular applications. Thus they can be significantlydifferent in spacing and color mixture, for example, on chromosomes thatare similar in general shape and size and which have similar bandingpatterns when conventional techniques are used. The size, shape andlabeling (e.g. color) of the hybridization bands produced by the probesof the present invention can be optimized to eliminate errors in machinescoring so that accurate automated aberration detection becomespossible. This optimized banding pattern will also greatly improvevisual chromosome classification and aberration detection.

The ease of recognition of specific translocation breakpoints can beimproved by using a reagent closely targeted to the region of the break.For example, a high complexity probe of this invention comprisingsequences that hybridize to both sides of the break on a chromosome canbe used. The portion of the probe that binds to one side of the breakcan be detected differently than that which binds to the other, forexample with different colors. In such a pattern, a normal chromosomewould have the different colored hybridization regions next to eachother, and such bands would appear close together. A break wouldseparate the probes to different chromosomes or result in chromosomalfragments, and could be visualized as much further apart on an average.

VII.B Biological Dosimetry

One approach to biological dosimetry is to measure frequencies ofstructurally aberrant chromosomes as an indication of the genetic damagesuffered by individuals exposed to potentially toxic agents. Numerousstudies have indicated the increase in structural aberration frequencieswith increasing exposure to ionizing radiation and other agents, whichare called clastogens. Dicentric chromosomes are most commonly scoredbecause their distinctive nature allows them to be scored rapidlywithout banding analysis. Rapid analysis is important because of the lowfrequency of such aberrations in individuals exposed at levels found inworkplaces (˜2×10⁻³ /cell). Unfortunately, dicentrics are not stablyretained so the measured dicentric frequency decreases with time afterexposure. Thus low level exposure over long periods of time does notresult in an elevated dicentric frequency because of the continuedclearance of these aberrations. Translocations are better aberrations toscore for such dosimetric studies because they are retained more or lessindefinitely. Thus, assessment of genetic damage can be made at timeslong after exposure. Translocations are not routinely scored forbiological dosimetry because the difficulty of recognizing them makesscoring sufficient cells for dosimetry logistically impossible.

The present invention eliminates this difficulty. Specifically,hybridization with a probe which substantially uniformly stains severalchromosomes (e.g. chromosomes 1, 2, 3 and 4) allows immediatemicroscopic identification in metaphase spreads of structuralaberrations involving these chromosomes. Normal chromosomes appearcompletely stained or unstained by the probe. Derivative chromosomesresulting from translocations between targeted and non-targetedchromosomes are recognized as being only partly stained, FIG. 4D. Suchpartially hybridized chromosomes can be immediately recognized eithervisually in the microscope or in an automated manner using computerassisted microscopy. Discrimination between translocations anddicentrics is facilitated by adding to the probe, sequences found at allof the chromosome centromeres. Detection of the centromeric componentsof the probe with a labeling means, for example color, different fromthat used to detect the rest of the probe elements allows readyidentification of the chromosome centromeres, which in turn facilitatesdiscrimination between dicentrics and translocations. This technologydramatically reduces the scoring effort required with previoustechniques so that it becomes feasible to examine tens of thousands ofmetaphase spreads as required for low level biological dosimetry.

VII.C. Prenatal Diagnosis

The most common aberrations found prenatally are trisomies involvingchromosomes 21 (Down syndrome), 18 (Edward syndrome) and 13 (Patausyndrome) and XO (Turner syndrome), XXY (Kleinfelter syndrome) and XYYdisease. Structural aberrations also occur. However, they are rare andtheir clinical significance is often uncertain. Thus, the importance ofdetecting these aberrations is questionable. Current techniques forobtaining fetal cells for conventional karyotyping, such as,amniocentesis and chorionic villus biopsy yield hundreds to thousands ofcells for analysis. These are usually grown in culture for 2 to 5 weeksto produce sufficient mitotic cells for cytogenetic analysis. Oncemetaphase spreads are prepared, they are analyzed by conventionalbanding analysis. Such a process can only be carried out by highlyskilled analysts and is time consuming so that the number of analysesthat can be reliably carried out by even the largest cytogeneticslaboratories is only a few thousand per year. As a result, prenatalcytogenetic analysis is usually limited to women whose children are athigh risk for genetic disease (e.g. to women over the age of 35).

The present invention overcomes these difficulties by allowing simple,rapid identification of common numerical chromosome aberrations ininterphase cells with no or minimal cell culture. Specifically, abnormalnumbers of chromosomes 21, 18, 13, X and Y can be detected in interphasenuclei by counting numbers of hybridization domains followinghybridization with probes specific for these chromosomes (or forimportant regions thereof such as 21q22 for Down syndrome). Ahybridization domain is a compact, distinct region over which theintensity of hybridization is high. An increased frequency of cellsshowing three domains (specifically to greater than 10%) for chromosomes21, 18 and 13 indicates the occurrence of Down, Edward and Patausyndromes, respectively. An increase in the number of cells showing asingle X-specific domain and no Y-specific domain followinghybridization with X-specific and Y-specific probes indicates theoccurrence of Turner syndrome. An increase in the frequency showing twoX-specific domains and one Y-specific domain indicates Kleinfeltersyndrome, and increase in the frequency of cells showing one X-specificdomain and two Y-specific domains indicates an XYY fetus. Domaincounting in interphase nuclei can be supplemented (or in some casesreplaced) by measurement of the intensity of hybridization using, forexample, quantitative fluorescence microscopy or flow cytometry, sincethe intensity of hybridization is approximately proportional to thenumber of target chromosomes for which the probe is specific. Numericalaberrations involving several chromosomes can be scored simultaneouslyby detecting the hybridization of the different chromosomes withdifferent labeling means, for example, different colors. Theseaberration detection procedures overcome the need for extensive cellculture required by procedures since all cells in the population can bescored. They eliminate the need for highly skilled analysts because ofthe simple, distinct nature of the hybridization signatures of numericalaberrations. Further, they are well suited to automated aberrationanalysis.

The fact that numerical aberrations can be detected in interphase nucleialso allows cytogenetic analysis of cells that normally cannot bestimulated into mitosis. Specifically, they allow analysis of fetalcells found in maternal peripheral blood. Such a feature is advantageousbecause it eliminates the need for invasive fetal cell sampling such asamniocentesis or chorionic villus biopsy.

As indicated in the Background, the reason such embryo-invasive methodsare necessary is that conventional karyotyping and banding analysisrequires metaphase chromosomes. At this time, there are no acceptedprocedures for culturing fetal cells separated from maternal blood toprovide a population of cells having metaphase chromosomes. In that thestaining reagents of this invention can be employed with interphasenuclei, a non-embryo-invasive method of karyotyping fetal chromosomes isprovided by this invention.

The first step in such a method is to separate fetal cells that havepassed through the placenta or that have been shed by the placenta intothe maternal blood. The incidence of fetal cells in the maternalbloodstream is very low, on the order of 10⁻⁴ to 10⁻⁶ cells/ml and quitevariable depending on the time of gestation; however, appropriatelymarked fetal cells may be distinguished from maternal cells andconcentrated, for example, with high speed cell sorting.

The presence of cells of a male fetus may be identified by a label, forexample a fluorescent tag, on a chromosome-specific staining reagent forthe Y chromosome. Cells that were apparently either lymphocytes orerythrocyte precursors that were separated from maternal blood wereshown to be Y-chromatin-positive. Zillacus et al., Scan. J. Haematol,15: 333 (1975); Parks and Herzenberg, Methods in Cell Biology, Vol. 10,pp. 277-295 (Academic Press, N.Y., 1982); and Siebers et al.,Humangenetik, 28: 273 (1975)!.

A preferred method of separating fetal cells from maternal blood is theuse of monoclonal antibodies which preferentially have affinity for somecomponent not present upon the maternal blood cells. Fetal cells may bedetected by paternal HLA (human leukocyte antigen) markers or by anantigen on the surface of fetal cells. Preferred immunochemicalprocedures to distinguish between fetal and maternal leukocytes on thebasis of differing HLA type use differences at the HLA-A2, -A3, and -B7loci, and further preferred at the -A2 locus. Further, first and secondtrimester fetal trophoblasts may be marked with antibody against theinternal cellular constituent cytokeratin which is not present inmaternal leukocytes. Exemplary monoclonal antibodies are described inthe following references.

Herzenberg et al., PNAS, 76: 1453 (1979), reports the isolation of fetalcells, apparently of lymphoid origin, from maternal blood byfluorescence activated cell sorting (FACS) wherein the separation wasbased on the detection of labeled antibody probes which bind HLA-A2negative cells in maternal blood. Male fetal cells separated in thatmanner were further identified by quinacrine staining of Y-chromatin.

Covone et al., Lancet, Oct. 13, 1984: 841, reported the recovery offetal trophoblasts from maternal blood by flow cytometry using amonoclonal antibody termed H315. Said monoclonal reportedly identifies aglycoprotein expressed on the surface of the human syncytiotrophoblastas well as other trophoblast cell populations, and that is absent fromperipheral blood cells.

Kawata et al., J. Exp. Med., 160: 653 (1984), discloses a method forisolating placental cell populations from suspensions of human placenta.The method uses coordinate two-color and light-scatter FACS analysis andsorting. Five different cell populations were isolated on the basis ofsize and quantitative differences in the coordinate expression of cellsurface antigens detected by monoclonal antibodies against an HLA-A, B,C monomorphic determinant (MB40.5) and against human trophoblasts(anti-Trop-1 and anti-Trop-2).

Loke and Butterworth, J. Cell Sci., 76: 189 (1985), describe twomonoclonal antibodies, 18B/A5 and 18A/C4, which are reactive with firsttrimester cytotrophoblasts and other fetal epithelial tissues includingsyncytiotrophoblasts.

A preferred monoclonal antibody to separate fetal cells from maternalblood for staining according to this invention is the anti-cytokeratinantibody Cam 5.2, which is commercially available from Becton-Dickinson(Franklin Lakes, N.J., USA).

Other preferred monoclonal antibodies for separating fetal cells frommaternal blood are those disclosed in co-pending, commonly owned U.S.patent application, U.S. Ser. No. 389,224, filed Aug. 3, 1989, entitled"Method for Isolating Fetal Cytotrophoblast Cells". See also: Fisher etal., J. Cell. Biol., 109 (2): 891-902 (1989)!. The monoclonal antibodiesdisclosed therein react specifically with antigen on first trimesterhuman cytotrophoblast cells, which fetal cells have the highestprobability of reaching the maternal circulation. Said application andarticle are herein specifically incorporated by reference. Briefly, thedisclosed monoclonal antibodies were raised by injection of test animalswith cytotrophoblast cells obtained from sections of the placental bed,that had been isolated by uterine aspiration. Antibodies raised weresubjected to several cytological screens to select for those antibodieswhich react with the cytotrophoblast stem cell layer of first trimesterchorionic villi.

Preferred monoclonal antibodies against such first trimestercytotrophoblast cells disclosed by Fisher et al. include monoclonalantibodies produced from the following hybridomas deposited at theAmerican Tyupe Culture Collection (ATCC; Rockville, Md., USA) under theBudapest Treaty:

    ______________________________________                                        Hybridoma    ATCC Accession #                                                 ______________________________________                                        J1D8         HB10096                                                          P1B5         HB10097                                                          ______________________________________                                    

Both hybridoma cultures were received by the ATCC on April 4, 1989 andreported viable thereby on Apr. 14, 1989.

Fisher et al. state that fetal cells isolated from maternal blood by useof said monoclonal antibodies are capable of replication in vitro.Therefore, fetal cells isolated by the method of Fisher et al., that is,first trimester fetal cytotrophoblasts, may provide fetal chromosomalmaterial that is both in metaphase and in interphase.

The fetal cells, preferably leukocytes and cytotrophoblasts, morepreferably cytotrophoblasts, once marked with an appropriate antibodyare then separated from the maternal cells either directly or bypreferably separating and concentrating said fetal cells by cell sortingor panning. For example, FACS may be used to separate fluorescentlylabeled fetal cells, or flow cytometry may be used.

The fetal cells once separated from the maternal blood can then bestained according to the methods of this invention with appropriatechromosome-specific staining reagents of this invention, preferablythose of particular importance for prenatal diagnosis. Preferredstaining reagents are those designed to detect aneuploidy, for example,trisomy of any of several chromosomes, including chromosome types 21,18, 13, X and Y and subregions on such chromosomes, such as, subregion21q22 on chromosome 21.

Preferably, a fetal sample for staining analysis according to thisinvention comprises at least 10 cells or nuclei, and more preferablyabout 100 cells or nuclei.

VII.D Tumor Cytogenetics

Numerous studies in recent years have revealed the existence ofstructural and numerical chromosome aberrations that are diagnostic forparticular disease phenotypes and that provide clues to the geneticnature of the disease itself. Prominent examples include the closeassociation between chronic myelogeneous leukemia and a translocationinvolving chromosome 9 and 22, the association of a deletion of aportion of 13q14 with retinoblastoma and the association of atranslocation involving chromosomes 8 and 14 with Burkitts lymphoma.Current progress in elucidating new tumor specific abnormalities islimited by the difficulty of producing representative, high qualitybanded metaphase spreads for cytogenetic analysis. These problems stemfrom the fact that many human tumors are difficult or impossible to growin culture. Thus, obtaining mitotic cells is usually difficult. Even ifthe cells can be grown in culture, there is the significant risk thatthe cells that do grow may not be representative of the tumorigenicpopulation. That difficulty also impedes the application of existinggenetic knowledge to clinical diagnosis and prognosis.

The present invention overcomes these limitations by allowing detectionof specific structural and numerical aberrations in interphase nuclei.These aberrations are detected as described supra. Hybridization withwhole chromosome probes will facilitate identification of previouslyunknown aberrations thereby allowing rapid development of newassociations between aberrations and disease phenotypes. As the geneticnature of specific malignancies becomes increasingly well known, theinterphase assays can be made increasingly specific by selectinghybridization probes targeted to the genetic lesion. Translocations atspecific sites on selected chromosomes can be detected by usinghybridization probes that closely flank the breakpoints. Use of theseprobes allows diagnosis of these specific disease phenotypes.Translocations may be detected in interphase because they bring togetherhybridization domains that are normally separated, or because theyseparate a hybridization domain into two, well separated domains. Inaddition, they may be used to follow the reduction and reemergence ofthe malignant cells during the course of therapy. Interphase analysis isparticularly important in such a application because of the small numberof cells that may be present and because they may be difficult orimpossible to stimulate into mitosis.

Duplications and deletions, processes involved in gene amplification andloss of heterozygosity, can also be detected in metaphase spreads andinterphase nuclei using the techniques of this invention. Such processesare implicated in an increasing number of different tumors.

The descriptions of the foregoing embodiments of the invention have beenpresented for purpose of illustration and description. They are notintended to be exhaustive or to limit the invention to the precise formdisclosed, and obviously many modifications and variations are possiblein light of the above teachings. The embodiments were chosen anddescribed in order to best explain the principles of the invention andits practical application to thereby enable others skilled in the art tobest utilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated. It isintended that the scope of the invention be defined by the claimsappended hereto.

All references cited herein are hereby incorporated by reference.

We claim:
 1. Nucleic acid probes for use in in situ hybridizationcomprising a heterogeneous mixture that contains labeled nucleic acidfragments that are substantially complementary to unique nucleic acidsegments, wherein said mixture comprises blocking nucleic acid havingnucleic acid fragments which are substantially complementary torepetitive segments in the labeled nucleic acid, produced by the processof:(a) obtaining chromosome-specific DNA fragments; (b) amplifying saidchromosome-specific DNA fragments; (c) labeling said chromosome-specificDNA fragments with a fluorescent or affinity label; and (d) addingsufficient blocking nucleic acid to prevent substantial binding of thelabeled nucleic acid sequences to repetitive nucleic acid sequences in atarget DNA.
 2. Nucleic acid probes according to claim 1, wherein saidstep (b) of amplifying said chromosome-specific DNA fragments isperformed by using a PCR process.
 3. Nucleic acid probes according toclaim 1, wherein said probes have a combined complexity of greater thanabout 40 kb.
 4. Nucleic acid probes according to claim 3, wherein saidprobes have a combined complexity of between about 40 and about 100 kb.5. Nucleic acid probes according to claim 1, wherein said probes have acombined complexity of greater than about 50 kb.
 6. Nucleic acid probesaccording to claim 1, wherein said chromosome-specific DNA fragments arelabeled by direct attachment of a fluorochrome.
 7. Nucleic acid probesaccording to claim 1, wherein said chromosome-specific DNA fragments areimmunochemically labeled.
 8. Nucleic acid probes according to claim 1,wherein said chromosome-specific DNA fragments are labeled with biotin.9. Nucleic acid probes according to claim 1, wherein saidchromosome-specific DNA fragments are labeled by direct attachment of anenzyme.
 10. Nucleic acid probes according to claim 1, wherein saidchromosome-specific DNA fragments are labeled by modification withN-acetoxy-N-2-acetylaminofluorene.
 11. Nucleic acid probes according toclaim 1, wherein said labeled nucleic acid fragments are complementaryto the total genomic complement of chromosomes, fragments complementaryto a single chromosome, fragments complementary to a subset ofchromosomes, or fragments complementary to a subregion of a singlechromosome.
 12. Nucleic acid probes according to claim 11, wherein saidlabeled nucleic acid fragments are selected from the nucleic acid ofnormal human chromosomes 1 through 22, X and Y.
 13. Nucleic acid probesaccording to claim 12, wherein said labeled nucleic acid fragments areselected from the nucleic acid of chromosome 21.